Biopolymers modified with superoxide dismutase mimics

ABSTRACT

This invention provides modified biopolymers comprising biopolymers attached to at least one non-proteinaceous catalyst capable of dismutating superoxide in the biological system or precursor ligand thereof. The invention further provides pharmaceutical compositions comprising the modified biopolymer and therapeutic methods comprising administering the modified biopolymer to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/580,007, filedMay 26, 2000, which claims priority from provisional application No.60/136,298 filed May 27, 1999, which are hereby incorporated byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

The present invention relates to biomaterials modified withnon-proteinaceous catalysts for the dismutation of superoxide, andprocesses for making such materials. This modification may be bycovalent conjugation, copolymerization, or admixture of thenon-proteinaceous catalysts with the biomaterial. The resulting modifiedbiomaterials exhibit a marked decrease in inflammatory response andsubsequent degradation when placed in contact with vertebrate biologicalsystems.

“Biomaterial” is a term given to a wide variety of materials which aregenerally considered appropriate for use in biological systems,including metals, polymers, biopolymers, and ceramics. Also included inthe term are composites of such materials, such as thepolymer-hydroxyapatite composite disclosed in U.S. Pat. No. 5,626,863.Biomaterials are used in a variety of medical and scientificapplications where a man-made implement comes into contact with livingtissue. Heart valves, stents, replacement joints, screws, pacemakerleads, blood vessel grafts, sutures and other implanted devicesconstitute one major use of biomaterials. Machines which handle bodilyfluids for return to the patient, such as heart/lung and hemodialysismachines, are another significant use for biomaterials.

Common metal alloy biomaterials used for implants include titaniumalloys, cobalt-chromium-molybdenum alloys,cobalt-chromium-tungsten-nickel alloys and non-magnetic stainless steels(300 series stainless steel). See U.S. Pat. No. 4,775,426. Titaniumalloys are frequently used for implants because they have excellentcorrosion resistance. However, they have inferior wear characteristicswhen compared with either cobalt-chromium-molybdenum alloys or 300series stainless steel. Cobalt-chromium-molybdenum alloys have about thesame tensile strength as the titanium alloys, but are generally lesscorrosion resistant. They also have the further disadvantage of beingdifficult to work. In contrast, the 300 series stainless steels weredeveloped to provide high-strength properties while maintainingworkability. These steels are, however, even less resistant to corrosionand hence more susceptible to corrosion fatigue. See U.S. Pat. No.4,718,908. Additional examples of biocompatible metals and alloysinclude tantalum, gold, platinum, iridium, silver, molybdenum, tungsten,inconel and nitinol. Because certain types of implants (artificialjoints, artificial bones or artificial tooth roots) require highstrength, metallic biomaterials have conventionally been used. However,as mentioned above, certain alloys corrode within the body and, as aresult, dissolved metallic ions can produce adverse effects on thesurrounding cells and can result in implant breakage.

In an attempt to solve this problem, ceramic biomaterials such asalumina have been used in high-stress applications such as in artificialknee joints. Ceramic biomaterials have an excellent affinity for bonetissue and generally do not corrode in the body. But when used under theload of walking or the like, they may not remain fixed to the bone. Inmany cases additional surgery is required to secure the loosenedimplant. This shortcoming led to the development of bioactive ceramicmaterials. Bioactive ceramics such as hydroxyapatite and tricalciumphosphate are composed of calcium and phosphate ions (the mainconstituents of bone) and are readily resorbed by bone tissue to becomechemically united with the bone. U.S. Pat. No. 5,397,362. However,bioactive ceramics such as hydroxyapatite and tricalcium phosphate arerelatively brittle and can fail under the loads in the human body. Thishas led in turn to the development of non-calcium phosphate bioactiveceramics with high strength. See U.S. Pat. No. 5,711,763. Additionalexamples of biocompatible ceramics include zirconia, silica, calcia,magnesia, and titania series materials, as well as the carbide seriesmaterials and the nitride series materials.

Polymeric biomaterials are desirable for implants because of theirchemical inertness and low friction properties. However, polymers usedin orthopedic devices such as hip and knee joints have a tendency forwear and build-up of fine debris, resulting in a painful inflammatoryresponse. Examples of biocompatible polymeric materials includesilicone, polyurethane, polyureaurethane, polyethylene teraphthalate,ultra high molecular weight polyethylene, polypropylene, polyester,polyamide, polycarbonate, polyorthoesters, polyesteramides,polysiloxane, polyolefin, polytetrafluoroethylene, polysulfones,polyanhydrides, polyalkylene oxide, polyvinyl halide, polyvinyledenehalide, acrylic, methacrylic, polyacrylonitrile, vinyl, polyphosphazene,polyethylene-co-acrylic acid, hydrogels and copolymers. Specificapplications include the use of polyethylene in hip and knee jointimplants and the use of hydrogels in ocular implants. See U.S. Pat. No.5,836,313. In addition to relatively inert polymeric materials discussedabove, certain medical applications require the use of biodegradablepolymers for use as sutures and pins for fracture fixation. Thesematerials serve as a temporary scaffold which is replaced by host tissueas they are degraded. See U.S. Pat. No. 5,766,618. Examples of suchbiodegradable polymers include polylactic acid, polyglycolic acid, andpolyparadioxanone.

In addition to wholly synthetic polymers, polymers which are naturallyproduced by organisms have been used in several medical applications.Such polymers, including polysaccharides such as chitin, cellulose andhyaluronic acid, and proteins such as fibroin, keratin, and collagen,offer unique physical properties in the biological environment, and arealso useful when a biodegradable polymer is required. In order to adaptthese polymers for certain uses, many have been chemically modified,such as chitosan and methyl cellulose. These polymers have found nichesin a variety of applications. Chitosan is often used to castsemi-permeable films, such as the dialysis membranes in U.S. Pat. No.5,885,609. Fibroin (silk protein) has been used as a support member intissue adhesive compositions, U.S. Pat. No. 5,817,303. Also, esters ofhyaluronic acid have been used to create bioabsorbable scaffolding forthe regrowth of nerve tissue, U.S. Pat. No. 5,879,359.

As is evident from the preceding paragraphs, individual biomaterialshave both desirable and undesirable characteristics. Thus, it is commonto create medical devices which are composites of various biocompatiblematerials in order to overcome these deficiencies. Examples of suchcomposite materials include: the implant material comprising glass fiberand polymer material disclosed in U.S. Pat. No. 5,013,323; thepolymeric-hydroxyapatite bone composite disclosed in U.S. Pat. No.5,766,618; the implant comprising a ceramic substrate, a thin layer ofglass on the substrate and a layer of calcium phosphate over the glassdisclosed in U.S. Pat. No. 5,397,362; and an implant material comprisingcarbon fibers in a matrix of fused polymeric microparticles. The diverseuses of biomaterials require a range of mechanical and physicalproperties for particular applications. As medical science advances,many applications will require new and diverse materials which can besafely and effectively used in biological systems.

Biomaterials, especially polymers, have been chemically modified inseveral ways in order to give them certain biological characteristics.For instance, thrombogenesis has posed a perennial problem for the useof biomaterials in hemodialysis membranes. In order to decreasethrombogenesis, hemodialysis fluid circuit materials have been modifiedby ionic complexation and interpenetration of heparin, U.S. Pat. No.5,885,609, and by graft copolymer techniques in which heparin is linkedto the backbone polymer by polyethylene oxide, Park, K. D., “Synthesisand Characterization of SPUU-PEO-Heparain Graft Copolymers”, J. Polymer.Sci., Vol. 20, p. 1725–37 (1991). Similarly, polymers containingincorporated drugs for elution into the body have been co-implanted withstents in order to prevent restenosis, U.S. Pat. No. 5,871,535.

Although most biomaterials in current use are considered non-toxic,implanted biomaterial devices are seen as foreign bodies by the immunesystem, and so elicit a well characterized inflammatory response. SeeGristina, A. G. “Implant Failure and the Immuno-IncompetentFibro-Inflammatory Zone” In “Clinical Orthopaedics and Related Research”(1994), No. 298, pp. 106–118. This response is evidenced by theincreased activity of macrophages, granulocytes, and neutrophils, whichattempt to remove the foreign object by the secretion of degradativeenzymes and free radicals like superoxide ion (0₂ ⁻) to inactivate ordecompose the foreign object. Woven dacron polyester, polyurethane,velcro, polyethylene, and polystyrene were shown to elicit superoxideproduction from neutrophils by Kaplan, S. S., et al,“Biomaterial-induced alterations of neutrophil superoxide production” In“Jour.Bio.Mat.Res.” (1992), Vol. 26, pp. 1039–1051. To a lesser extent,polysulfone/carbon fiber and polyetherketoneketone/carbon fibercomposites were shown to elicit a superoxide response by Moore, R., etal, “A comparison of the inflammatory potential of particulates derivedfrom two composite materials” In “Jour.Bio.Mat.Res.” (1997), Vol. 34,pp. 137–147. Hydroxyapatite, tricalcium phosphate, andaluminum-calcium-phosphorous oxide bioceramics were shown to be degradedby macrophages by Ross, L., et al, “The Effect of HA, TCP and AlcapBioceramic Capsules on the Viability of Human Monocyte and MonocyteDerived Macrophages” in “Bio.Sci.Inst.” (1996), Vol. 32, pp. 71–79.Similarly, cobalt-chrome alloy beads were degraded by neutrophils in astudy by Shanbhag, A., et al, “Decreased neutrophil respiratory burst onexposure to cobalt-chrome alloy and polystyrene in vitro” In“Jour.Bio.Mat.Res.” (1992), Vol. 26, 2, pp. 185–195. Even biomaterialswhich have been modified to present biologically acceptable molecules,such as heparin, have been found to elicit an inflammatory response,Borowiec, J. W., et al, “Biomaterial-Dependent Blood Activation DuringSimulated Extracorporeal Circulation: a Study of Heparin-Coated andUncoated Circuits”, Thorac. Cardiovasc. Surgeon 45 (1997) 295–301. Inaddition, chemical modification has posed several difficulties. Becauseof the unique chemical characteristics of each biomaterial and bioactivemolecule, covalent linkage of the desired bioactive molecule to thebiomaterial is not always possible. In addition, the activity of manybioactive molecules, especially proteins, is diminished or extinguishedwhen anchored to a solid substrate. Finally, the fact that manybiologically active substances are heat liable has prevented their usewith biomaterials that are molded or worked at high temperatures.

The impact of continual attempts by the organism to degrade biomaterialimplants can lead to increased morbidity and device failure. In the caseof polyurethane pacemaker lead wire coatings, this results in polymerdegradation and steady loss of function. In the use of syntheticvascular grafts, this results in persistent thrombosis, improperhealing, and restenosis. As mentioned above, orthopedic devices such aship and knee joints have a tendency for wear and build-up of fine debrisresulting in a painful inflammatory response. In addition, thesurrounding tissue does not properly heal and integrate into theprosthetic device, leading to device loosening and opportunisticbacterial infections. It has been proposed by many researchers thatchronic inflammation at the site of implantation leads to the exhaustionof the macrophages and neutrophils, and an inability to fight offinfection.

Superoxide anions are normally removed in biological systems by theformation of hydrogen peroxide and oxygen in the following reaction(hereinafter referred to as dismutation):O₂ ⁻+O₂ ⁻+2H⁺→O₂+H₂O₂This reaction is catalyzed in vivo by the ubiquitous superoxidedismutase enzyme. Several non-proteinaceous catalysts which mimic thissuperoxide dismutating activity have been discovered. A particularlyeffective family of non-proteinaceous catalysts for the dismutation ofsuperoxide consists of the manganese(II), manganese(III), iron(II) oriron(III) complexes of nitrogen-containing fifteen-membered macrocyclicligands which catalyze the conversion of superoxide into oxygen andhydrogen peroxide, described in U.S. Pat. Nos. 5,874,421 and 5,637,578,all of which are incorporated herein by reference. See also Weiss, R.H., et al, “Manganese(II)-Based Superoxide Dismutase Mimetics: RationalDrug Design of Artificial Enzymes”, (1996) Drugs of the Future 21,383–389; and Riley, D. P., et al, “Rational Design of Synthetic Enzymesand Their Potential Utility as Human Pharmaceuticals” (1997) in CatTech,I, 41. These mimics of superoxide dismutase have been shown to have avariety of therapeutic effects, including anti-inflammatory activity.See Weiss, R. H., et al, “Therapeutic Aspects of Manganese (II)-BasedSuperoxide Dismutase Mimics” In “Inorganic Chemistry in Medicine”,(Farrell, N., Ed.), Royal Society of Chemistry, in Press; Weiss, R. H.,et al, “Manganese-Based Superoxide Dismutase Mimics: Design, Discoveryand Pharmacologic Efficacies” (1995) In “The Oxygen Paradox (Davies, K.J. A., and Ursini, F., Eds.) pp. 641–651, CLEUP University Press,Padova, Italy; Weiss, R. H., et al, “Manganese-Based SuperoxideDismutase Mimetic Inhibit Neutrophil Infiltration In Vitro”,J.Biol.Chem., 271, 26149 (1996); and Hardy, M. M., et al, “SuperoxideDismutase Mimetics Inhibit Neutrophil-Mediated Human Aortic EndothelialCell Injury In Vitro”, (1994) J.Biol.Chem. 269, 18535–18540. Othernon-proteinaceous catalysts which have been shown to have superoxidedismutating activity are the salen-transition metal cation complexes,described in U.S. Pat. No. 5,696,109, and complexes of porphyrins withiron and manganese cations.

SUMMARY OF THE INVENTION

Applicants have discovered that the modification of biomaterials withnon-proteinaceous catalysts for the dismutation of superoxide greatlyimproves the biomaterial's resistance to degradation and reduces theinflammatory response. Thus, the present invention is directed tobiomaterials which have been modified with non-proteinaceous catalystsfor the dismutation of superoxide, or precursor ligands ofnon-proteinaceous catalysts for the dismutation of superoxide.

The present invention is directed to biomaterials which have beenmodified with non-proteinaceous catalysts for the dismutation ofsuperoxide, or precursor ligands of a non-proteinaceous catalyst for thedismutation of superoxide, by utilizing methods of physical association,such as surface covalent conjugation, copolymerization, and physicaladmixing. The present invention is also directed to biomaterialsmodified with non-proteinaceous catalysts for the dismutation ofsuperoxide wherein one or more of these methods has been used to modifythe biomaterial.

A variety of biomaterials are appropriate for modification in thepresent invention. Because the non-proteinaceous catalysts for thedismutation of superoxide are suitable for use in a range of methods forphysically associating the catalyst with the biomaterial, almost anybiomaterial may be modified according to the present invention. Thebiomaterial to be modified may be any biologically compatible metal,ceramic, polymer, biopolymer, biologically derived material, or acomposite thereof. Thus, the present invention is further directedtowards any of the above biomaterials modified with non-proteinaceouscatalysts for the dismutation of superoxide.

As previously mentioned, the non-proteinaceous catalysts for thedismutation of superoxide for use in the present invention comprise anorganic ligand and a transition metal cation. Particularly preferredcatalysts are manganese and iron chelates of pentaazacyclopentadecanecompounds (hereinafter referred to as “PACPeD catalysts”). Also suitablefor use in the present invention are the salen complexes of manganeseand iron disclosed in U.S. Pat. No. 5,696,109, and iron or manganeseporphyrins, such as Mn^(III) tetrakis(4-N-methylpyridyl)porphyrin,Mn^(III) tetrakis-o-(4-N-methylisonicotinamidophenyl)porphyrin, Mn^(III)tetrakis(4-N-N-N-trimethylanilinium)porphyrin, Mn^(III)tetrakis(1-methyl-4-pyridyl)porphyrin, Mn^(III) tetrakis(4-benzoicacid)porphyrin, Mn^(III)octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin, Fe^(III)tetrakis(4-N-methylpyridyl)porphyrin, and Fe^(III)tetrakis-o-(4-N-methylisonicotinamidophenyl)porphyrin. Thesenon-proteinaceous catalysts for the dismutation of superoxide alsopreferably contain a reactive moiety when the methods of surfacecovalent conjugation or copolymerization are used to modify thebiomaterial. Thus, the present invention is directed to biomaterialswhich have been modified with any of the above non-proteinaceouscatalysts for the dismutation of superoxide. In addition, as sometimesit is advantageous to add the chelated transition metal ion after thebiomaterial has been modified, the present invention is also directed tobiomaterials which have been modified with the precursor ligand of anyof the above non-proteinaceous catalysts.

The present invention is also directed to processes for producingbiomaterials modified by surface covalent conjugation with at least onenon-proteinaceous catalyst for the dismutation of superoxide or at leastone precursor ligand of a non-proteinaceous catalyst for the dismutationof superoxide, the process comprising:

-   -   a. providing at least one reactive functional group on a surface        of the biomaterial to be modified;    -   b. providing at least one complementary reactive functional        group on the non-proteinaceous catalyst for the dismutation of        superoxide or on the precursor ligand; and    -   c. conjugating the non-proteinaceous catalyst for the        dismutation of superoxide or the precursor ligand with the        surface of the biomaterial through at least one covalent bond.

-   The non-proteinaceous catalyst for the dismutation of superoxide or    the precursor ligand can be covalently bound directly to the surface    of the biomaterial, or bound to the surface through a linker    molecule. Thus, the present invention is also directed to the above    process further comprising providing a bi-functional linker    molecule.

The present invention is also directed to a process for producing abiomaterial modified by co-polymerization with at least onenon-proteinaceous catalyst for the dismutation of superoxide or at leaston ligand precursor of a non-proteinaceous catalyst for the dismutationof superoxide, the process comprising:

-   -   a. providing at least one monomer;    -   b. providing at least one non-proteinaceous catalyst for the        dismutation of superoxide or at least one ligand precursor of a        non-proteinaceous catalyst for the dismutation of superoxide        containing at least one functional group capable of reaction        with the monomer and also containing at least one functional        group capable of propagation of the polymerization reaction,    -   c. copolymerizing the monomers and the non-proteinaceous        catalyst for the dismutation of superoxide or the ligand        precursor in a polymerization reaction.

The present invention is also directed to a process for producing abiomaterial modified by admixture with at least one non-proteinaceouscatalyst for the dismutation of superoxide or a precursor ligand of anon-proteinaceous catalyst for the dismutation of superoxide, theprocess comprising:

-   -   a. providing at least one unmodified biomaterial;    -   b. providing at least one non-proteinaceous catalyst for the        dismutation of superoxide or at least one ligand precursor of a        non-proteinaceous catalyst for the dismutation of superoxide;        and    -   c. admixing the unmodified biomaterial and the non-proteinaceous        catalyst for the dismutation of superoxide or the ligand        precursor.

In addition, the present invention is also directed to a novel method ofsynthesizing PACPeD catalysts by using manganese or other transitionmetal ions as a template for cyclization the ligand.

The present invention is also directed to a biocompatible articlecomprising a biomaterial modified with at least one non-proteinaceouscatalyst for the dismutation of superoxide or a ligand precursor of anon-proteinaceous catalyst for the dismutation of superoxide, whereinthe catalyst or ligand precursor is presented on a surface of thearticle. The invention is also directed to the use of the biomaterialsof the present invention in a stent, a vascular graft fabric, a nervegrowth channel, a cardiac lead wire, or other medical devices forimplantation in or contact with the body or bodily fluids.

BRIEF DESCRIPTION OF DRAWINGS AND DEFINITIONS

Drawings

FIG. 1: An electron micrograph of the surface of a control disk ofpoly(etherurethane urea) which has not been implanted.

FIG. 2: An electron micrograph of the surface of a control disk ofpoly(etherurethane urea) (not conjugated with a non-proteinaceouscatalyst for the dismutation of superoxide) which has been implanted ina rat for 28 days.

FIG. 3: An electron micrograph of the surface of a poly(etherurethaneurea) disc which has been conjugated with Compound 43 and which has beenimplanted in a rat for 28 days.

FIG. 4: A comparison of capsules formed around polypropylene fiberswhich have been implanted into a rat. A) a control fiber, made ofpolypropylene which has not been admixed with a non-proteinaceouscatalyst for the dismutation of superoxide; B) a fiber made ofpolypropylene which has been admixed with Compound 54, 2% by weight.

FIG. 5: A comparison of capsules formed around disks of polyethylenewhich have been implanted in a rat for 3 days. A) control disk, notconjugated with a non-proteinaceous catalyst; B) a disk conjugated withCompound 43, 0.06% by weight; C) a disc conjugated with Compound 43,1.1% by weight.

FIG. 6: A comparison of capsules formed around disks of polyethylenewhich have been implanted in a rat for 28 days. A) control disk, notconjugated with a non-proteinaceous catalyst; B) a disk conjugated withCompound 43, 0.06% by weight; C) a disc conjugated with Compound 43,1.1% by weight.

FIG. 7: A graphical comparison of the capsule thickness and number ofgiant cells in the capsule for polyethylene disks conjugated withCompound 43, 0.06% by weight, and polyethylene disks conjugated withCompound 43, 1.1% by weight, after implantation for 28 days.

FIG. 8: A comparison of capsules formed around disks ofpoly(etherurethane urea) which have been implanted in a rat for 28 days.A) control disk, not conjugated with a non-proteinaceous catalyst; B) adisk conjugated with Compound 43, 0.6% by weight; C) a disc conjugatedwith Compound 43, 3.0% by weight.

FIG. 9: A comparison of capsules formed around disks of tantalum whichhave been implanted in a rat for 3 days. A) control disk, conjugatedonly with the silyl linker; B) a disk conjugated with Compound 43 viathe silyl linker.

FIG. 10: A comparison of capsules formed around disks of tantalum whichhave been implanted in a rat for 28 days. A) control disk, conjugatedonly with the silyl linker; B) a disk conjugated with Compound 43 viathe silyl linker.

FIG. 11: A drawing of the unwound wire used to make the stent of Example26.

FIG. 12: A close up of the bends and “eyes” in the wire of FIG. 11.

FIG. 13: A side view drawing of the helically wound stent, fullyexpanded.

FIG. 14: A cross-section of the helically wound stent.

FIG. 15: A side view drawing of the helically wound stent, compressed.

FIG. 16: A detailed view of the helically wound stent, showing the angleof the helix (∃) and the angle between the zig-zags of the stent wire(∀).

Definitions

As utilized herein, the term “biomaterial” includes any generallynon-toxic material commonly used in applications where contact withbiological systems is expected. Examples of biomaterials include: metalssuch as stainless steel, tantalum, titanium, nitinol, gold, platinum,inconel, iridium, silver, molybdenum, tungsten, nickel, chromium,vanadium, and alloys comprising any of the foregoing metals and alloys;ceramics such as hydroxyapatite, tricalcium phosphate, andaluminum-calcium-phosphorus oxide; polymers such as polyurethane,polyureaurethane, polyalkylene glycols, polyethylene teraphthalate,ultra high molecular weight polyethylenes, polypropylene, polyesters,polyamides, polycarbonates, polyorthoesters, polyesteramides,polysiloxanes, polyolefins, polytetrafluoroethylenes, polysulfones,polyanhydrides, polyalkylene oxides, polyvinyl halides, polyvinyledenehalides, acrylics, methacrylics, polyacrylonitriles, polyvinyls,polyphosphazenes, polyethylene-co-acrylic acid, silicones, blockcopolymers of any of the foregoing polymers, random copolymers of any ofthe foregoing polymers, graft copolymers of any of the foregoingpolymers, crosslinked polymers of any of the foregoing polymers,hydrogels, and mixtures of any of the foregoing polymers; biopolymerssuch as chitin, chitosan, cellulose, methyl cellulose, hyaluronic acid,keratin, fibroin, collagen, elastin, and saccharide polymers;biologically derived materials such as fixed tissues, and composites ofsuch materials. “Biocompatible” articles are fabricated out ofbiomaterials. As used herein, the term “biomaterial” is not meant toencompass drugs and biologically active molecules such as steroids,di-saccharides and short chain polysaccharides, fatty acids, aminoacids, antibodies, vitamins, lipids, phospholipids, phosphates,phosphonates, nucleic acids, enzymes, enzyme substrates, enzymeinhibitors, or enzyme receptor substrates.

The term “non-proteinaceous catalysts for the dismutation of superoxide”means a low-molecular-weight catalyst for the conversion of superoxideanions into hydrogen peroxide and molecular oxygen. These catalystscommonly consist of an organic ligand and a chelated transition metalion, preferably manganese or iron. The term may include catalystscontaining short-chain polypeptides (under 15 amino acids), ormacrocyclic structures derived from amino acids, as the organic ligand.The term explicitly excludes a superoxide dismutase enzyme obtained fromany species.

The term “precursor ligand” means the organic ligand of anon-proteinaceous catalyst for the dismutation of superoxide without thechelated transition metal cation.

The term “biopolymer” means a polymer which can be produced in a livingsystem or synthetically out of amino acids, saccharides, or othertypical biological monomers. The term also encompasses derivatives ofthese biological polymers. Examples of biopolymers include chitin,chitosan, cellulose, methyl cellulose, hyaluronic acid, keratin,fibroin, collagen, and elastin.

The term “biologically derived material” means biological tissue whichhas been chemically modified for implantation into a new host, such asfixed heart valves and blood vessels.

The term “modification” means any method by which a physical associationmay be effected between a biomaterial and a non-proteinaceous catalystfor the dismutation of superoxide, whereby the non-proteinaceouscatalyst becomes integrated into or onto the biomaterial. Modificationmay be effected by surface covalent conjugation, copolymerization,admixture, or by other methods. When modification is achieved byadmixture, it is understood that the non-proteinaceous catalyst is inthe same phase as at least a part of the biomaterial that is modified.

The term “surface covalent conjugation” means that the non-proteinaceouscatalyst is bound through at least one covalent bond to the surface of abiomaterial. The term encompasses conjugation via a direct covalent bondbetween the non-proteinaceous catalyst and the surface, as well as anindirect bond which includes a linker molecule between thenon-proteinaceous catalyst and the surface of the biomaterial.

The term “linker” means any molecule with at least two functional groupswhich can be used to “link” one molecule to another. Examples of linkersinclude low molecular weight polyethylene glycol, hexamethyldi(imidi)-isocyanate, silyl chloride, and polyglycine.

The term “copolymerization” means that the non-proteinaceous catalyst iscopolymerized with the monomer which forms the biomaterial, and thusintegrated into the polymer chain of the modified biomaterial.

The term “inflammatory response” means that the material elicits theinflammation of the surrounding tissues and the production ofdegradative enzymes and reactive molecular species when exposed tobiological systems.

The term “substituted” means that the described moiety has one or moreof the following substituents:

-   -   (1) —NR₃₀R₃₁ wherein R₃₀ and R₃₁ are independently selected from        hydrogen, alkyl, aryl or aralkyl; or R₃₀ is hydrogen, alkyl,        aryl or aralkyl and R₃₁ is selected from the group consisting of        —NR₃₂R₃₃, —OH, —OR₃₄,        wherein R₃₂ and R₃₃ are independently hydrogen, alkyl, aryl or        acyl, R₃₄ is alkyl, aryl or alkaryl, Z′ is hydrogen, alkyl,        aryl, alkaryl, —OR₃₄, —SR₃₄ or —NR₄₀R₄₁. R₃₇ is alkyl, aryl or        alkaryl, X′ is oxygen or sulfur, and R₃₈ and R₃₉ are        independently selected from hydrogen, alkyl, or aryl;    -   (2) —SR₄₂ wherein R₄₂ is hydrogen, alkyl, aryl, alkaryl, —SR₃₄,        —NR₃₂R₃₃,        wherein R₄₃ is —OH, —OR₃₄ or —NR₃₂R₃₃, and A and B are        independently —OR₃₄, —SR₃₄ or —NR₃₂R₃₃    -   (3) wherein x is 1 or 2, and R₄₄ is halide, alkyl, aryl,        alkaryl, —OH, —OR₃₄ or —NR₃₂R₃₃;    -   (4) —OR₄₅ wherein R₄₅ is hydrogen, alkyl, aryl, alkaryl,        —NR₃₂R₃₃,        wherein D and E are independently —OR₃₄ or —NR₃₂R₃₃;        wherein R₄₆ is halide, —OH, —SH, —OR₃₄, —SR₃₄ or —NR₃₂R₃₃;    -   (6) amine oxides of the formula        provided R₃₀ and R₃₁ are not hydrogen;    -   (7)        wherein F and G are independently —OH, —SH, —OR₃₄, —SR₃₄ or        —NR₃₂R₃₃;    -   (8) —O—(—(CH₂)_(a)—O)_(b)—R₁₀ wherein R₁₀ is hydrogen or alkyl,        and a and b are integers independently selected from 1+6;    -   (9) halogen, cyano, nitro or azido; or    -   (10) aryl, heteroaryl, alkynyl, or alkenyl.        Alkyl, aryl and alkaryl groups on the substituents of the        above-defined alkyl groups may contain one or more additional        substituents, but are preferably unsubstituted.

The term “functional group” means a group capable of reacting withanother functional group to form a covalent bond. Functional groupspreferably used in the present invention include acid halide (XCO—wherein X=Cl, F, Br, I), amino (H₂N—), isocyanate (OCN—), mercapto(HS—), glycidyl (H₂COCH—), carboxyl (HOCO—), hydroxy (HO—), andchloromethyl (ClH₂C—), silyl or silyl chloride, and substituted orunsubstituted alkenyl, alkynyl, aryl, and heteroaryl.

The term “alkyl”, alone or in combination, means a straight-chain orbranched-chain alkyl radical containing from 1 to about 22 carbon atoms,preferably from about 1 to about 18 carbon atoms, and most preferablyfrom about 1 to about 12 carbon atoms. Examples of such radicalsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl,octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl andeicosyl.

The term “alkenyl”, alone or in combination, means an alkyl radicalhaving one or more double bonds. Examples of such alkenyl radicalsinclude, but are not limited to, ethenyl, propenyl, 1-butenyl,cis-2-butenyl, trans-2-butenyl, iso-butylenyl, cis-2-pentenyl,trans-2-pentenyl, 3-methyl-1-butenyl, 2,3-dimethyl-2-butenyl,1-pentenyl, 1-hexenyl, 1-octenyl, decenyl, dodecenyl, tetradecenyl,hexadecenyl, cis- and trans-9-octadecenyl, 1,3-pentadienyl,2,4-pentadienyl, 2,3-pentadienyl, 1,3-hexadienyl, 2,4-hexadienyl,5,8,11,14-eicosatetraenyl, and 9,12,15-octadecatrienyl.

The term “alkynyl”, alone or in combination, means an alkyl radicalhaving one or more triple bonds. Examples of such alkynyl groupsinclude, but are not limited to, ethynyl, propynyl (propargyl),1-butynyl, 1-octynyl, 9-octadecynyl, 1,3-pentadiynyl, 2,4-pentadiynyl,1,3-hexadiynyl, and 2,4-hexadiynyl.

The term “cycloalkyl”, alone or in combination means a cycloalkylradical containing from 3 to about 10, preferably from 3 to about 8, andmost preferably from 3 to about 6, carbon atoms. Examples of suchcycloalkyl radicals include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, andperhydronaphthyl.

The term “cycloalkylalkyl” means an alkyl radical as defined above whichis substituted by a cycloalkyl radical as defined above. Examples ofcycloalkylalkyl radicals include, but are not limited to,cyclohexylmethyl, cyclopentylmethyl, (4-isopropylcyclohexyl)methyl,(4-t-butyl-cyclohexyl)methyl, 3-cyclohexylpropyl,2-cyclohexylmethylpentyl, 3-cyclopentylmethylhexyl,1-(4-neopentylcyclohexyl)methylhexyl, and1-(4-isopropylcyclohexyl)methylheptyl.

The term “cycloalkylcycloalkyl” means a cycloalkyl radical as definedabove which is substituted by another cycloalkyl radical as definedabove. Examples of cycloalkylcycloalkyl radicals include, but are notlimited to, cyclohexylcyclopentyl and cyclohexylcyclohexyl.

The term “cycloalkenyl”, alone or in combination, means a cycloalkylradical having one or more double bonds. Examples of cycloalkenylradicals include, but are not limited to, cyclopentenyl, cyclohexenyl,cyclooctenyl, cyclopentadienyl, cyclohexadienyl and cyclooctadienyl.

The term “cycloalkenylalkyl” means an alkyl radical as defined abovewhich is substituted by a cycloalkenyl radical as defined above.Examples of cycloalkenylalkyl radicals include, but are not limited to,2-cyclohexen-1-ylmethyl, 1-cyclopenten-1-ylmethyl,2-(1-cyclohexen-1-yl)ethyl, 3-(1-cyclopenten-1-yl)propyl,1-(1-cyclohexen-1-ylmethyl)pentyl, 1-(1-cyclopenten-1-yl)hexyl,6-(1-cyclohexen-1-yl)hexyl, 1-(1-cyclopenten-1-yl)nonyl and1-(1-cyclohexen-1-yl)nonyl.

The terms “alkylcycloalkyl” and “alkenylcycloalkyl” mean a cycloalkylradical as defined above which is substituted by an alkyl or alkenylradical as defined above. Examples of alkylcycloalkyl andalkenylcycloalkyl radicals include, but are not limited to,2-ethylcyclobutyl, 1-methylcyclopentyl, 1-hexylcyclopentyl,1-methylcyclohexyl, 1-(9-octadecenyl)cyclopentyl and1-(9-octadecenyl)cyclohexyl.

The terms “alkylcycloalkenyl” and “alkenylcycloalkenyl” means acycloalkenyl radical as defined above which is substituted by an alkylor alkenyl radical as defined above. Examples of alkylcycloalkenyl andalkenylcycloalkenyl radicals include, but are not limited to,1-methyl-2-cyclopentyl, 1-hexyl-2-cyclopentenyl, 1-ethyl-2-cyclohexenyl,1-butyl-2-cyclohexenyl, 1-(9-octadecenyl)-2-cyclohexenyl and1-(2-pentenyl)-2-cyclohexenyl.

The term “aryl”, alone or in combination, means a phenyl or naphthylradical which optionally carries one or more substituents selected fromalkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl,alkoxy, halogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy,ether, trifluoromethyl and the like, such as phenyl, p-tolyl,4-methoxyphenyl, 4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl,4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, and the like.

The term “aralkyl”, alone or in combination, means an alkyl orcycloalkyl radical as defined above in which one hydrogen atom isreplaced by an aryl radical as defined above, such as benzyl,2-phenylethyl, and the like.

The term “heterocyclic” means ring structures containing at least oneother kind of atom, in addition to carbon, in the ring. The most commonof the other kinds of atoms include nitrogen, oxygen and sulfur.Examples of heterocyclics include, but are not limited to, pyrrolidinyl,piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl,thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl,indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl,benzoxadiazolyl, benzothiadiazolyl, triazolyl and tetrazolyl groups.

The term “saturated, partially saturated or unsaturated cyclic” meansfused ring structures in which 2 carbons of the ring are also part ofthe fifteen-membered macrocyclic ligand. The ring structure can contain3 to 20 carbon atoms, preferably 5 to 10 carbon atoms, and can alsocontain one or more other kinds of atoms in addition to carbon. The mostcommon of the other kinds of atoms include nitrogen, oxygen and sulfur.The ring structure can also contain more than one ring.

The term “saturated, partially saturated or unsaturated ring structure”means a ring structure in which one carbon of the ring is also part ofthe fifteen-membered macrocyclic ligand. The ring structure can contain3 to 20, preferably 5 to 10, carbon atoms and can also contain nitrogen,oxygen and/or sulfur atoms.

The term “nitrogen containing heterocycle” means ring structures inwhich 2 carbons and a nitrogen of the ring are also part of thefifteen-membered macrocyclic ligand. The ring structure can contain 2 to20, preferably 4 to 10, carbon atoms, can be substituted orunsubstituted, partially or fully unsaturated or saturated, and can alsocontain nitrogen, oxygen and/or sulfur atoms in the portion of the ringwhich is not also part of the fifteen-membered macrocyclic ligand.

The term “organic acid anion” refers to carboxylic acid anions havingfrom about 1 to about 18 carbon atoms.

The term “halide” means chloride, floride, iodide, or bromide.

As used herein, “R” groups means all of the R groups attached to thecarbon atoms of the macrocycle, i.e., R, R′, R₁, R′₁, R₂, R′₂, R₃, R′₃,R₄, R′₄, R₅, R′₅, R₆, R′₆, R′₇, R₈, R′₈, R₉.

All references cited herein are explicitly incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns novel modified biomaterials and methodsfor the production of such materials. Prior to applicants' invention, itwas not known that non-proteinaceous catalysts for the dismutation ofsuperoxide could be immobilized on the surface of a biomaterial andstill retain their catalytic function and exhibit an anti-inflammatoryeffect. However, applicants have found that these catalysts can beefficaciously immobilized on biomaterial surfaces and still retainsuperoxide dismutating ability, as shown by Example 23. Applicants havealso found that these modified biomaterials have greatly improveddurability and decreased inflammatory response when exposed tobiological systems, such as the rat model in Examples 21 and 22.

Biomaterials and Non-Proteinaceous Catalysts for the Dismutation ofSuperoxide for Use in the Present Invention

A variety of biomaterials are appropriate for modification in thepresent invention. The biomaterial to be modified can be anybiologically compatible metal, ceramic, polymer, biopolymer, or acomposite thereof. Metals suitable for use in the present inventioninclude stainless steel, tantalum, titanium, nitinol, gold, platinum,inconel, iridium, silver, molybdenum, tungsten, nickel, chromium,vanadium, and alloys comprising any of the foregoing metals and alloys.Ceramics suitable for use in the present invention includehydroxyapatite, tricalcium phosphate, and aluminum-calcium-phosphorusoxide. Polymers suitable for use in the present invention includepolyurethane, polyureaurethane, polyalkylene glycols, polyethyleneteraphthalate, ultra high molecular weight polyethylenes, polypropylene,polyesters, polyamides, polycarbonates, polyorthoesters,polyesteramides, polysiloxanes, polyolefins, polytetrafluoroethylenes,polysulfones, polyanhydrides, polyalkylene oxides, polyvinyl halides,polyvinyledene halides, acrylics, methacrylics, polyacrylonitriles,polyvinyls, polyphosphazenes, polyethylene-co-acrylic acid, silicones,block copolymers of any of the foregoing polymers, random copolymers ofany of the foregoing polymers, graft copolymers of any of the foregoingpolymers, crosslinked polymers of any of the foregoing polymers,hydrogels, and mixtures of any of the foregoing polymers. Biopolymerssuitable for use in the present invention are chitin, chitosan,cellulose, methyl cellulose, hyaluronic acid, keratin, fibroin,collagen, elastin, and saccharide polymers. Composite materials whichmay be used in the present invention comprise a relatively inelasticphase such as carbon, hydroxy apatite, tricalcium phosphate, silicates,ceramics, or metals, and a relatively elastic phase such as a polymer orbiopolymer.

Where the method used to modify the biomaterial is surface covalentconjugation, the unmodified biomaterial should contain, or be chemicallyderivatized to contain, a reactive moiety. Preferred reactive moietiesinclude acid halide (XCO— wherein X=Cl, F, Br, I), amino (H₂N—),isocyanate (OCN—), mercapto (HS—), glycidyl (H₂COCH—), carboxyl (HOCO—),hydroxy (HO—), and chloromethyl (ClH₂C—), silyl or silyl chloride, andsubstituted or unsubstituted alkenyl, alkynyl, aryl, and heteroarylmoieties.

Applicants have discovered that these compounds, especially thepreferred pentaaza non-proteinaceous catalysts, will survive a widerange of chemical reactions and processing conditions including extremechemical and thermal conditions. Particularly, the PACPeD catalysts havebeen demonstrated by the applicants to be stable at temperatures up toabout 350EC, and at pH of about 4. Additionally, the PACPeD's aresoluble in a wide range of solvents, including water, methanol, ethanol,methylene chloride, DMSO, DMF, and DMAC, and are partially soluble intoluene and acetonitrile. By adding polar or non-polar substituents atthe R group positions on the PACPeD or other non-proteinaceouscatalysts, applicants have improved their solubility in specificsolvents for particular reactions, and for use with particularbiomaterials. As illustrated by Table 1 below, several reactivefunctional groups may be added as pendant moieties without detrimentallyaffecting the catalyst's superoxide dismutating ability.

The non-proteinaceous catalysts for the dismutation of superoxide foruse in the present invention preferably comprise an organic ligand and atransition metal cation. Particularly preferred catalysts are manganeseand iron chelates of pentaazacyclopentadecane compounds, which can berepresented by the following formula:

wherein M is a cation of a transition metal, preferably manganese oriron; wherein R, R′, R₁, R′₁, R₂, R′₂, R₃, R′₃, R₄, R′₄, R₅, R′₅, R₆,R′₆, R₇, R′₇, R₈, R′₈, R₉, and R′₉ independently represent hydrogen, orsubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl,alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl,alkenylcycloalkenyl, heterocyclic, aryl and aralkyl radicals; R₁ or R′₁and R₂ or R′₂, R₃ or R′₃ and R₄ or R′₄, R₅ or R′₅ and R₆ or R′₆, R₇ orR′₇ and R₈ or R′₈, and R₉ or R′₉ and R or R′ together with the carbonatoms to which they are attached independently form a substituted orunsubstituted, saturated, partially saturated or unsaturated cyclic orheterocyclic having 3 to 20 carbon atoms; R or R′and R₁ or R′₁, R₂ orR′₂ and R₃ or R′₃, R₄ or R′₄ and R₅ or R′₅, R₆ or R′₆ and R₇ or R′₇, andR₈ or R′₈ and R₉ or R′₉ together with the carbon atoms to which they areattached independently form a substituted or unsubstituted nitrogencontaining heterocycle having 2 to 20 carbon atoms, provided that whenthe nitrogen containing heterocycle is an aromatic heterocycle whichdoes not contain a hydrogen attached to the nitrogen, the hydrogenattached to the nitrogen as shown in the above formula, which nitrogenis also in the macrocyclic ligand or complex, and the R groups attachedto the included carbon atoms of the macrocycle are absent; R and R′, R₁and R′₁, R₂ and R′₂, R₃ and R′₃, R₄ and R′₄, R₅ and R′₅, R₆ and R′₆, R₇and R′₇, R₈ and R′₈, and R₉ and R′₉, together with the carbon atom towhich they are attached independently form a saturated, partiallysaturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbonatoms; and one of R, R′, R₁, R′₁, R₂, R′₂, R₃, R′₃, R₄, R′₄, R₅, R′₅,R₆, R′₆, R₇, R′₇, R₈, R′₈, R₉, and R′₉ together with a different one ofR, R′, R₁, R′₁, R₂, R′₂, R₃, R′₃, R₄, R′₄, R₅, R′₅, R₆, R′₆, R₇, R′₇,R₈, R′₈, R₉, and R′₉ which is attached to a different carbon atom in themacrocyclic ligand may be bound to form a strap represented by theformula—(CH₂)_(x)-M-(CH₂)_(w)-L-(CH₂)_(z)—I—(CH₂)_(y)—wherein w, x, y and z independently are integers from 0 to 10 and M, Land J are independently selected from the group consisting of alkyl,alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl,aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide,phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol,carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy,silaza and combinations thereof; and combinations thereof. Thus, thePACPeD's useful in the present invention can have any combinations ofsubstituted or unsubstituted R groups, saturated, partially saturated orunsaturated cyclics, ring structures, nitrogen containing heterocycles,or straps as defined above.

X, Y and Z represent suitable ligands or charge-neutralizing anionswhich are derived from any monodentate or polydentate coordinatingligand or ligand system or the corresponding anion thereof (for examplebenzoic acid or benzoate anion, phenol or phenoxide anion, alcohol oralkoxide anion). X, Y and Z are independently selected from the groupconsisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen,peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino,arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides,hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide,cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, arylnitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido,alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, arylsulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenicacid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylicacid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, arylthiol thiocarboxylic acid, alkyl carboxylic acid (such as acetic acid,trifluoroacetic acid, oxalic acid), aryl carboxylic acid (such asbenzoic acid, phthalic acid), urea, alkyl urea, aryl urea, alkyl arylurea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea,sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite,hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide,aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphinesulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkylphosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, arylphosphinic acid, alkyl phosphinous acid, aryl phosphinous acid,phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate,hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, arylguanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkylaryl carbamate, alkyl thiocarbamate aryl thiocarbamate, alkyl arylthiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryldithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate,chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite,tetrahalomanganate, tetrafluoroborate, hexafluorophosphate,hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate,tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate,citrate, ascorbate, saccharinate, amino acid, hydroxamic acid,thiotosylate, and anions of ion exchange resins. The preferred ligandsfrom which X, Y and Z are selected include halide, organic acid, nitrateand bicarbonate anions.

The “R” groups attached to the carbon atoms of the macrocycle can be inthe axial or equatorial position relative to the macrocycle. When the“R” group is other than hydrogen or when two adjacent “R” groups, i.e.,on adjacent carbon atoms, together with the carbon atoms to which theyare attached form a saturated, partially saturated or unsaturated cyclicor a nitrogen containing heterocycle, or when two R groups on the samecarbon atom together with the carbon atom to which they are attachedform a saturated, partially saturated or unsaturated ring structure, itis preferred that at least some of the “R” groups are in the equatorialposition for reasons of improved activity and stability. This isparticularly true when the complex contains more than one “R” groupwhich is not hydrogen.

Where the modification of the biomaterial is effected by the surfacecovalent conjugation or copolymerization with the unmodifiedbiomaterial, it is preferred that the PACPeD contain a pendant reactivemoiety. This reactive moiety may be on a “R” group, a cyclic, aheterocyclic, a nitrogen containing heterocyclic, or a strap structureas described above. Preferred moieties on the non-proteinaceous catalystfor use in the present invention include of amino (—NH₂), carboxyl(—OCOH), isocyanate (—NCO), mercapto (—SH), hydroxy (—OH), silylchloride (—SiCl₂), acid halide (—OCX wherein X=Cl, F, Br, I), halide (—Xwherein X=Cl, F, Br, I), glycidyl (—HCOCH₂), and substituted orunsubstituted alkenyl, alkynyl, and aryl moieties.

Preferred PACPeD's for modification of biomaterials compounds are thosewherein at least one “R” group contains a reactive functional group, andthose wherein at least one, of R or R′and R₁ or R′₁, R₂ or R′₂ and R₃ orR′₃, R₄ or R′₄ and R₅ or R′₅, R₆ or R′₆ and R₇ or R′₇, and R₈ or R′₈ andR₉ or R′₉ together with the carbon atoms to which they are attached arebound to form a nitrogen containing heterocycle having 2 to 20 carbonatoms and all the remaining “R” groups are independently selected fromhydrogen, saturated, partially saturated or unsaturated cyclic or alkylgroups. Examples of PACPeD catalysts useful in making the modifiedbiomaterials of the invention include, but are not limited to, thefollowing compounds:

TABLE 1 MOL. k_(cat) pH _(kcat) pH COMPOUND WT. 7.4 8.1

341.19 4.13 2.24

431.31 7.21 2.57

403.26 1.00

379.23 1.75

411.77 3.82 3.90

447.31 6.99 3.83

501.37 2.00 1.58

584.39 5.95 5.90

423.22 2.71 1.68

491.32 2.68 2.68

452.37 4.79 2.85

610.42 10.20 5.39

383.27 1.63

506.46 7.58 3.84

795.95 2.41 0.77

481.41 2.48 1.97

449.37 12.60 4.09

463.40 15.00 4.00

437.36 8.48 4.08

485.70 3.29 0.93

599.67 2.93 1.29

494.63 11.40 5.03

461.29 6.61 3.47

493.38 2.55 2.55

724.39 4.04 2.34

479.40 10.12 3.47

525.47 4.83 2.50

411.56

454.10 2.86 2.02

409.22 0.20 0.20

480.43 2.97 2.91

681.70 1.74 1.43

629.44 7.27 4.08

685.55 2.70 2.78

827.76 4.38 2.87

877.72 0.63 0.49

549.49 3.08

483.39 1.64 1.19

535.46 3.89 2.32

511.44 90.00 11.00

511.44 1.57 0.41

517.83 1.18 0.98

679.76 1.02 0.84

587.51 2.99 0.95

563.52

537.48 2.16

562.28 1.68

614.52

641.50 1.31

573.53 3.97 1.14

537.02 3.01

579.56 2.68

Activity of the non-proteinaceous catalysts for the dismutation ofsuperoxide can be demonstrated using the stopped-flow kinetic analysistechnique as described in Example 24, and in Riley, D. P., Rivers, W..J. and Weiss, R. H., “Stopped-Flow Kinetic Analysis for MonitoringSuperoxide Decay in Aqueous Systems,” Anal. Biochem., 196, 344–349(1991), which is incorporated by reference herein. Stopped-flow kineticanalysis is an accurate and direct method for quantitatively monitoringthe decay rates of superoxide in water. The stopped-flow kineticanalysis is suitable for screening compounds for SOD activity andactivity of the compounds or complexes of the present invention, asshown by stopped-flow analysis, correlate to usefulness in the modifiedbiomaterials and processes of the present invention. The catalyticconstants given for the exemplary compounds in the table above weredetermined using this method.

As can be observed from the table, a wide variety of PACPeD's withsuperoxide dismutating activity may be readily synthesized. Generally,the transition metal center of the catalyst is thought to be the activesite of catalysis, wherein the manganese or iron ion cycles between the(II) and (III) states. Thus, as long as the redox potential of the ionis in a range in which superoxide anion can reduce the oxidized metaland protonated superoxide can oxidize the reduced metal, and sterichindrance of the approach of the superoxide anion is minimal, thecatalyst will function with a kcat of about 10⁶ to 10⁸.

Without limiting themselves to any particular theory, applicants proposethat the mechanism described in Riley, et al., 1999, is a reasonableapproximation of how the PACPeD catalysts dismutate superoxide. In orderfor the complex to exhibit superoxide dismutase activity, the ligandshould be able to fold into a conformation that allows the stabilizationof an octahedral complex between the superoxide anion and the fivenitrogens of the ligand ring. If a compound contains several conjugateddouble bonds within the main 15-membered ring of the ligand, which holdthe ring in a rigid conformation, the compound would not be expected toexhibit catalytic activity. R groups which are coordinated with thetransition metal ion freeze the conformation of the ligand, and would beexpected to be poor catalysts. Large, highly electronegative groupspendant on the macrocycle would also sterically hinder the necessaryconformational change. The lack of functionality in these types ofPACPeD derivatives would not be unexpected by one of ordinary skill inthe art. Specifically, one of skill in the art would avoid materiallychanging the flexibility of the PACPeD by adding many large groups whichwould cause steric hindrance, or placing too many double bonds into themain PACPeD ring. This effect would also be present in certain geometricarrangements of smaller R groups which constrain the complex to a rigid,planar geometry. Those particular compounds which do not exhibitsuperoxide dismutase activity should not be used to modify thebiomaterials of the present invention.

Given these examples and guidelines, one of ordinary skill would be ableto choose a PACPeD catalyst for use in the present invention which wouldcontain any required functional group, while still retaining superoxidedismutating activity. The PACPeD catalysts described above may beproduced by the methods disclosed in U.S. Pat. No. 5,610,293. However,it is preferred that the PACPeD catalysts used in the present inventionbe synthesized by the template method, diagramed below. This synthesismethod is advantageous over previously disclosed methods in thatcyclization yields utilizing the template method are usually about 90%,as compared to about 20% with previous methods. Several diamines arecommercially available as starting materials, or a diamine may besynthesized. The diamine is reacted with titryl chloride in anhydrousmethylene chloride at 0EC and allowed to warm to room temperatureovernight, with stirring. The product is then combined with glyoxal inmethanol and stirred for 16 hours. The glyoxal bisimine product is thenreduced with a borohydride in THF. If a non-symmetrical product isdesired, two diamines may be used as starting materials. In addition, asubstituted glyoxal may be used if groups pendant from the macrocycleopposite the pyridine are desired (R₅ and R₄) Commercially availabletetraamines may also be used in place of the reduced glyoxal bisimine.After reduction of the glyoxal bisimine, the product is combined with a2,6 dicarbonyl substituted pyridine, such as 2,6, dicarboxaldyhydepyridine or 2,6 diacetyl pyridine, and a salt of manganese or iron underbasic conditions. The transition metal ion serves as a template topromote cyclization of the substituted pyridine and the tetraamine.Several 2,6 dicarbonyl substituted pyridines are available commercially,allowing for the facile production of a variety of ligands with groupspendant from the macrocycle proximal to the pyridine (R₂ and R₃).Additionally, pyridines with additional substitutions (R₆, R₇ and R₈)may also be used. After cyclization, the product is reduced withammonium formate and a palladium catalyst over a period of 3–4 days. Inaddition to the “R” substitutions, “R′” groups may also be substitutedat the same carbons. “R” and “R′” groups may be any of those indicatedabove. The process may be varied according to principles well known toone of ordinary skill in the art in order to accommodate variousstarting materials.

Although the bisimine produced in the template cyclization reaction stepabove may be reduced by more conventional means using hydrogen gas, itis preferred that the bisimine be reduced with ammonium formate in thepresence of a palladium catalyst, as illustrated in Example 6. Thisprocess offers the advantages of increased safety and high reductionefficiency.

The PACPeD's useful in the present invention can possess one or moreasymmetric carbon atoms and are thus capable of existing in the form ofoptical isomers as well as in the form of racemic or nonracemic mixturesthereof. The optical isomers can be obtained by resolution of theracemic mixtures according to conventional processes, for example byformation of diastereoisomeric salts by treatment with an opticallyactive acid. Examples of appropriate acids are tartaric,diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric andcamphorsulfonic acid and then separation of the mixture ofdiastereoisomers by crystallization followed by liberation of theoptically active bases from these salts. A different process forseparation of optical isomers involves the use of a chiralchromatography column optimally chosen to maximize the separation of theenantiomers. Still another available method involves synthesis ofcovalent diastereoisomeric molecules by reacting one or more secondaryamine group(s) of the compounds of the invention with an optically pureacid in an activated form or an optically pure isocyanate. Thesynthesized diastereoisomers can be separated by conventional means suchas chromatography, distillation, crystallization or sublimation, andthen hydrolyzed to deliver the enantiomerically pure ligand. Theoptically active compounds of the invention can likewise be obtained byutilizing optically active starting materials, such as natural aminoacids.

Also suitable for use in the present invention, but less preferred thanthe PACPeD's, are the salen complexes of manganese and iron disclosed inU.S. Pat. No. 5,696,109, here incorporated by reference. The term “salencomplex” means a ligand complex with the general formula:

wherein M is a transition metal ion, preferably Mn; A is an anion,typically Cl; and n is either 0, 1, or 2. X₁, X₂, X₃ and X₄ areindependently selected from the group consisting of hydrogen, silyls,arlyls, aryls, arylalkyls, primary alkyls, secondary alkyls, tertiaryalkyls, alkoxys, aryloxys, aminos, quaternary amines, heteroatoms, andhydrogen; typically X₁ and X₃ are from the same functional group,usually hydrogen, quaternary amine, or tertiary butyl, and X₂ and X₄ aretypically hydrogen. Y₁, Y₂, Y₃, Y₄, Y₅, and Y₆ are independentlyselected from the group consisting of hydrogen, halides, alkyls, aryls,arylalkyls, silyl groups, aminos, alkyls or aryls bearing heteroatoms;aryloxys, alkoxys, and halide; preferably, Y₁ and Y₄ are alkoxy, halide,or amino groups. Typically, Y₁ and Y₄ are the same. R₁, R₂, R₃ and R₄are independently selected from the group consisting of H, CH₃, C₂ H₅,C₆H₅, O-benzyl, primary alkyls, fatty acid esters, substitutedalkoxyaryls, heteroatom-bearing aromatic groups, arylalkyls, secondaryalkyls, and tertiary alkyls. Methods of synthesizing these salencomplexes are also disclosed in U.S. Pat. No. 5,696,109.

Iron or manganese porphyrins, such as , such as Mn^(III)tetrakis(4-N-methylpyridyl)porphyrin, Mn^(III)tetrakis-o-(4-N-methylisonicotinamidophenyl)porphyrin, Mn^(III)tetrakis(4-N-N-N-trimethylanilinium)porphyrin, Mn^(III)tetrakis(1-methyl-4-pyridyl)porphyrin, Mn^(III) tetrakis(4-benzoicacid)porphyrin, Mn^(III)octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin, Fe^(III)tetrakis(4-N-methylpyridyl)porphyrin, and Fe^(III)tetrakis-o-(4-N-methylisonicotinamidophenyl)porphyrin. may also be usedin the present invention. The catalytic activities and methods ofpurifying or synthesizing these porphyrins are well known in the organicchemistry arts.

The salen and porphyrin non-proteinaceous catalysts for the dismutationof superoxide also preferably contain a reactive moiety, as describedabove, when the methods of surface covalent conjugation orcopolymerization are used to modify the biomaterial.

In general, the non-proteinaceous catalysts for the dismutation ofsuperoxide used in the present invention are very stable underconditions of high heat, acid or basic conditions, and in a wide varietyof solvents. However, under extreme reaction conditions the chelatedtransition metal ion will dissociate from the non-proteinaceouscatalyst. Thus, when extreme reaction conditions are necessary to modifythe biomaterial, it is preferable to modify the biomaterial with aprecursor ligand of the non-proteinaceous catalyst for the dismutationof superoxide, and then afterwards react the modified biomaterial with acompound containing the appropriate transition metal in order to producea biomaterial modified with an active non-proteinaceous catalyst for thedismutation of superoxide. For instance, when a PACPeD catalyst is usedunder reaction conditions of pH <4, the strategy of modifying thebiomaterial with the ligand should be used. This strategy isdemonstrated in Example 19. Therefore, when the term non-proteinaceouscatalyst for the dismutation of superoxide is used in thisspecification, the reader should assume that, where appropriate, theprecursor ligand will be used in the modification of the biomaterial,and that the transition metal cation necessary for activity may be addedat a later point in time. Conditions where this approach would beappropriate may be readily determined by one of ordinary skill in thechemical arts.

Choice of Method of Modification

As previously described, the biomaterials of the present invention maybe modified by the diverse methods of surface covalent conjugation,copolymerization, or admixture. The methods of surface covalentconjugation and copolymerization use covalent bonds in order tophysically associate the non-proteinaceous catalyst for the dismutationof superoxide with the biomaterial. This creates a very stable physicalassociation which preserves the superoxide dismutating activity of themodified biomaterial. In contrast, non-covalent forces create thephysical association between the biomaterial and the non-proteinaceouscatalysts for the dismutation of superoxide when the technique ofphysical admixture is used. These non-covalent forces may be weak Vander Wal's forces, or they may be stronger ionic bonding or hydrophobicinteraction forces. Although ionic or hydrophobic interactions betweenthe non-proteinaceous catalyst and the biomaterial will prevent elutionof the non-proteinaceous catalyst to some degree, the catalyst willstill be lost from the biomaterial over time when the biomaterial isexposed to biological tissues or fluids. Thus, it is usually preferredthat the methods of covalent surface conjugation or copolymerization beused to modify biomaterials which will be exposed to biological systemsfor prolonged periods of time. However, uses may arise where the elutionof non-proteinaceous catalysts for the dismutation of superoxide intothe tissues surrounding an article comprising the modified biomaterialmay be desirable. In this case, the use of biomaterials modified by thephysical admixture method would be appropriate.

When composite materials are used, it may be necessary to utilize avariety of modification techniques. For instance, in a biomaterialcomposed of hydroxyapatite and polyethylene, a non-proteinaceouscatalyst may be admixed with the hydroxyapatite phase of the composite,and another copolymerized with the polyethylene phase of the composites.The two composites may then be joined together into a fully modifiedcomposite biomaterial. Similarly, a composite material which utilizescarbon fiber and polypropylene could be made using a copolymerizedpolypropylene and a surface covalently conjugated carbon fiber. Theflexibility in the production of modified biomaterials offered by theprocesses of the invention allows for the use of several diversematerials in a device while increasing its durability and decreasing theinflammatory response to the device.

Generally, it is preferred that the non-proteinaceous catalyst bepresent in an amount of about 0.001 to 25 weight percent. It is morepreferable that the catalyst be present in an amount of about 0.01 to 10weight percent. It is most preferable that the catalyst be present in anamount of about 0.05 to 5 weight percent. However, the amount of thenon-proteinaceous catalyst to be used in modifying the biomaterial willdepend on several factors, including the characteristics of thecatalyst, the characteristics of the biomaterial, and the method ofmodification used. As is evident from the chart above, the catalyticactivity of the non-proteinaceous catalysts for use in the presentinvention may vary over several orders of magnitude. Thus, less of themore efficient catalysts will be needed to obtain the same protectiveeffects. Also, some biomaterials are more inflammatory than others.Thus, a greater amount of catalyst should be used with thesebiomaterials in order to counteract the strong inflammatory foreign bodyresponse that they provoke. In addition, the amount of catalyst used tomodify the biomaterial should not be so high as to significantly alterthe mechanical characteristics of the biomaterial. Because a covalentlyconjugated catalyst is concentrated at the surface of the biomaterialused in a device, almost all of the catalyst will interact with thebiological environment. Conversely, because an admixed or copolymerizedcatalyst is dispersed throughout the biomaterial, less of the catalystwill be available to interact with the biological environment at thesurface of the biomaterial. Thus, when the catalyst is covalentlyconjugated to the surface of the biomaterial, less catalyst will beneeded than if the catalyst is admixed or copolymerized with thebiomaterial. Given the above considerations, the person of ordinaryskill in the art would be able to choose a proper amount ofnon-proteinaceous catalyst to use in the present invention in order toachieve the desired reduction in the inflammatory response anddegradation.

It is to be understood that although the non-proteinaceous catalystsused in the following processes are usually referred to in the singular,multiple catalysts may be used in any of these processes. One ofordinary skill in the art will easily be able to choose complementarycatalysts for such modified biomaterials. In addition, although notspecifically enumerated herein, the combination of the biomaterialmodification techniques of the present invention with other biomaterialmodification techniques, such as heparin coating, is contemplated withinthe present invention.

Modification by Surface Covalent Conjugation

The general process for producing a biomaterial modified by surfacecovalent conjugation with at least one non-proteinaceous catalyst forthe dismutation of superoxide or at least one precursor ligand of anon-proteinaceous catalyst for the dismutation of superoxide, comprises:

-   -   a. providing at least one reactive functional group on a surface        of the biomaterial to be modified;    -   b. providing at least one complementary reactive functional        group on the non-proteinaceous catalyst for the dismutation of        superoxide or on the precursor ligand; and    -   c. conjugating the non-proteinaceous catalyst for the        dismutation of superoxide or the precursor ligand with the        surface of the biomaterial through at least one covalent bond.

This process may be effected by a photo-chemical reaction, or any of anumber of conjugating reactions known in the art, such as condensation,esterification, oxidative, exchange, or substitution reactions.Preferred conjugation reactions for use in the present invention do notinvolve extreme reaction conditions, such as a temperature above about375EC, or pH less than about 4. In addition, it is preferred that theconjugation reaction not produce a covalent bond that is readily cleavedby common enzymes found in biological systems. Usually, it is desirablefor the non-proteinaceous catalyst to have only one complementaryfunctional group. However, in cases where crosslinking of thebiomaterial is desired, such as in hydrogels, poly-functional-groupcatalysts may be used. Care should be taken, however, to choosefunctional groups which will not allow the non-proteinaceous catalyst toself-polymerize, as this will decrease the efficiency of the conjugationreaction. Likewise, multiple non-proteinaceous catalysts may be used tomodify the biomaterial, although complementary functional groups whichallow avoid inter-catalyst conjugations would not be preferred.

The non-proteinaceous catalyst for the dismutation of superoxide or theprecursor ligand may be covalently bound directly to the surface of thebiomaterial, or bound to the surface through a linker molecule. Wherethe non-proteinaceous catalyst and the surface of the biomaterial aredirectly conjugated, the reactive functional group and the complementaryreactive functional group will form a covalent bond in the conjugationreaction. For instance, poly(ethyleneterephthalate) may be hydrolyzed tocarboxyl functional groups. Compound 43 may then be reacted with thederivatized polymer to form the amide bond, as illustrated in Example 7.Examples H and E also illustrate a direct surface covalent conjugation.Further suggestions for reactive groups to use in of direct conjugationmay be found in U.S. Pat. No. 5,830,539, herein incorporated byreference. Several exemplary paired functional groups are given in Table2:

TABLE 2 Non- proteinaceous Catalyst Substrate (R) (SODm) Group GroupResulting Linkage SODm-NH₂ R—N═C═O

SODm-NH₂

SODm-NH₂

SODm-NH₂

R—CH═NH-SODm SODm-NH₂

SODm-NH₂ R—N═C═S

SODm-OH R—N═C═O

SODm-OH

SODm-OH

SODm-OH R—N═C═S

SODm-OH

SODm-OH R—Si—(OCH₃)₃ R—Si—(O-SODm)₃

R—OH

R—N═C═O

R—NH₂

When a linker molecule is used, the above process further comprisesproviding at least one linker capable of reacting with both the reactivefunctional group on a surface of the biomaterial to be modified and thecomplementary reactive functional group on the non-proteinaceouscatalyst for the dismutation of superoxide or the precursor ligand.During the conjugation process, the reactive functional group on thesurface of the article and the complementary reactive functional groupon the non-proteinaceous catalyst for the dismutation of superoxide forma covalent bond with the linker. This process may occur all in one step,or in a series of steps. For instance, in a two step process, a carboxylfunctionalized polymer, such as a hydrolyzed poly(ethyleneterephthalate)polymer (“PET”) could first be reacted with a (Gly)₁₂ linker in an amidereaction. Then, after removal of excess linker, the PET-glycine linkercould react with an amino PACPeD such as Compound 43 to form apolymer-glycine linker-Compound 43 modified biomaterial. Alternately,the hydrolyzed PET could be linked with a low molecular weight PEG to acarboxyl PACPeD such as Compound 52 by an ester reaction in a singlestep. Linkers suitable for use in this process include polysaccharides,polyalkylene glycols, polypeptides, polyaldehydes, and silyl groups.Silyl groups are particularly useful in conjugating non-proteinaceouscatalysts with metal biomaterials. Examples of linkers and functionalgroups which are useful in the present invention may be found in U.S.Pat. Nos. 5,877,263 and 5,861,032. Persons of ordinary skill in thechemical arts will be able to determine an appropriate linker andnon-proteinaceous catalyst for conjugation to any biomaterial, includingmetals, ceramics, polymers, biopolymers, and various phases ofcomposites.

This method of modification may be used with an article which is alreadyin its final form, or may be used with parts of an article before finalassembly. In addition, this method is useful for modifying thin stockmaterials which will be used in the later manufacture of a device, suchas polymer or chitosan films, or fibers which will be woven into fabricsfor vascular grafts. This method is also useful for modifying diversematerials in a single step with one non-proteinaceous catalyst. Forinstance, a tantalum component which has been reacted with a silyllinker, as in Example 13, and a poly(ethyleneterephthalate) componentwhich has been hydrolyzed, as in Example 7, may be assembled into afinal device. Then, Compound 43 could be reacted with the entire articleto modify the surface of both materials in a single step.

Modification by Copolymerization

Biomaterials may also be modified according to the present invention byco-polymerization with a non-proteinaceous catalyst for the dismutationof superoxide or the ligand precursor of a non-proteinaceous catalystfor the dismutation of superoxide. This process, inc general, comprises:

-   -   a. providing at least one monomer;    -   b. providing at least one least one non-proteinaceous catalyst        for the dismutation of superoxide or at least one ligand        precursor of a non-proteinaceous catalyst for the dismutation of        superoxide containing at least one functional group capable of        reaction with the monomer and also containing at least one        functional group capable of propagation of the polymerization        reaction,    -   c. copolymerizing the monomers and the non-proteinaceous        catalyst for the dismutation of superoxide or the ligand        precursor in a polymerization reaction.

The copolymerization technique is advantageous for the modification ofpolymers and synthetic biopolymers with non-proteinaceous catalysts forthe dismutation of superoxide. However, it is preferred that this methodbe used with polymers whose polymerization reaction occurs attemperatures less than about 375EC, and pH greater than about 4. If thepolymerization reaction is carried out at a pH less than 4, a ligandprecursor of the non-proteinaceous catalysts for the dismutation ofsuperoxide should be used. Monomers useful in this process includealkylenes, vinyls, vinyl halides, vinyledenes, diacids, acid amines,diols, alcohol acids, alcohol amines, diamines, ureas, urethanes,phthalates, carbonic acids, orthoesters, esteramines, siloxanes,phosphazenes, olefins, alkylene halides, alkylene oxides, acrylic acids,sulfones, anhydrides, acrylonitriles, saccharides, and amino acids.

As demonstrated previously, the non-proteinaceous catalysts for thedismutation of superoxide used in the present invention may besynthesized with any functional group necessary to react with the any ofthese monomers. In order to prevent the termination of thepolymerization reaction, it is necessary that the non-proteinaceouscatalyst also contain a polymerization propagation functional group.Often, this will be another functional group identical to the firstfunctional group, as in the diamine PACPeD Compound 16. This catalyst iscopolymerized with polyureaurethane in Example 16. However, as when thepolymerization reaction involves a vinyl reaction, the reactive andpropagative functional groups may be the same, such as in the acryloylderivatized Compound 53. Copolymerization of this catalyst with acrylicor methacrylic is shown in Example 17. Example 18 also illustrates themodification of biomaterials by copolymerization with non-proteinaceouscatalysts.

Biomaterials modified by copolymerization have several advantages.First, the non-proteinaceous catalysts for the dismutation of superoxideare covalently bound to the modified biomaterial, preventingdissociation of the catalysts and a loss of function. Second, themodification of the material is continuous throughout the biomaterial,allowing for continuous protection by the catalyst if the exteriorsurface of the material is by mechanical or chemical degradation. Third,the material can be melted and re-formed into any useful article aftermodification, provided that the polymer melts below about 375EC.Alternatively, wet-spinning or solvent casting may be used to makearticles from these modified polymer biomaterials. These characteristicsmake the modified polymer biomaterials produced by this process aversatile tool for various medical device applications.

Modification by Admixture

The biomaterials of the present invention may also be modified byadmixture with at least one non-proteinaceous catalyst for thedismutation of superoxide or a precursor ligand of a non-proteinaceouscatalyst for the dismutation of superoxide. The general processcomprises:

-   -   a. providing at least one unmodified biomaterial;    -   b. providing at least one non-proteinaceous catalyst for the        dismutation of superoxide or at least one ligand precursor of a        non-proteinaceous catalyst for the dismutation of superoxide;        and    -   c. admixing the unmodified biomaterial and the non-proteinaceous        catalyst for the dismutation of superoxide or the ligand        precursor.

Biomaterials modified according to this process preferably form asolution with the non-proteinaceous catalyst or ligand, although a :m tonm-sized particle mixture is also contemplated by the present invention.The above admixture process may involve heating the constituents inorder to melt at least one unmodified biomaterial constituent. Forinstance, the PACPeD catalyst Compound 38 can be mixed with meltedpolypropylene at 250EC, as in Example 20. Many other polymerbiomaterials melt below 300EC, such as polyethylene,poly(ethyleneterephthalate) and polyamides, and would be especiallysuitable for use in this melted admixture technique. After admixing, themelted modified biomaterial may be injection or extrusion molded, orspun. Temperatures above about 375EC should not be used, however, asdecomposition of the catalyst may result.

Thus, metals, ceramics, and high-melt polymers should not be melted foradmixture. Rather, a solvent in which at least one unmodifiedbiomaterial and the non-proteinaceous catalyst for the dismutation ofsuperoxide or the ligand precursor are soluble may be used when admixingthese constituents. As noted above, the PACPeD catalysts are soluble inseveral common solvents. If the solvent method is used, the processpreferably further comprises removing the solvent after admixing.Methods suitable for removing a solvent used in the present inventioninclude evaporation and membrane filtration, although care should betaken so that the membrane filter size will retain the non-proteinaceouscatalyst. As with the copolymerized modified biomaterials, the admixedmodified biomaterials may be wet spun or solution cast.

More hydrophobic or hydrophilic groups may be added to thenon-proteinaceous catalyst in order to change its solubilitycharacteristics. Likewise, the non-proteinaceous catalysts may besynthesized with specific pendant groups in order to have a particularaffinity for the modified biomaterial. Usually this is accomplished bychoosing the non-proteinaceous catalyst used in the admixture process sothat ionic or hydrophobic interactions will occur between the catalystsand the modified biomaterial. For instance, the negatively chargedcarboxyl group of Compound 52 would have an affinity for the positivelycharged calcium ions in a hydroxyapatite ceramic matrix. Similarly, theadded cyclohexyl group of Compound 47, as well as the lack of pendantpolar groups, would help this catalyst to integrate into polyethylene.Thus, by increasing the affinity of the non-proteinaceous catalyst forthe biomaterial, one can help to prevent the dissociation of thecatalyst from the modified biomaterial.

Uses of the Modified Biomaterials

The biomaterials of the present invention show greatly improveddurability and decreased inflammatory response when interacting withbiological systems. Thus, these biomaterials modified withnon-proteinaceous catalysts for the dismutation of superoxide are idealfor use in devices for implantation or the handling of bodily fluids.Since the non-proteinaceous catalysts for the dismutation of superoxideare not consumed during the dismutation reaction, they may retain theiractivity indefinitely. The biocompatible article can be an articlewhere, during its intended use, at least a portion of the articlecomprising the modified biomaterial is implanted within a mammal. Forinstance, one such application would be coating pacemaker lead wires asdescribed in U.S. Pat. No. 5,851,227 with the modified polyureaurethaneof Example 16. These improved lead wires are believed to be more durablein the body, and thus prevent the device failure which is often seenwith conventional polyurethane coated wires. Similarly, a modifiedpolyester, such as in Example 19, could be used to spin fibers forvascular graft fabric as described in U.S. Pat. No. 5,824,047. Graftsmade using this fabric are believed to heal faster, as less inflammationwould be caused by the biomaterial. Similarly, the modifiedpolypropylene tested in Example 22 could be used to make surgicalsutures. The biocompatible article may also be one where, during itsintended use, the surface comprising the modified biomaterial is exposedto biological fluids, such as blood or lymph. For instance, a surfacecovalently conjugated chitosan film would be ideal for use as a membranematerial in heart-lung machines which oxygenate and circulate bloodduring bypass operations. The copolymerized poly(etherurethane urea) ofExample 16 would be useful in manufacturing the direct mechanicalbi-ventricular cardiac assist device of U.S. Pat. No. 5,749,839. Use ofthese biomaterials in tissue engineering devices, such as scaffoldings,would be another application.

The various methods of modifying biomaterials provided by the inventionallow for a wide range of practical applications. For instance, inmanufacturing stents for use in angioplasty procedures, one would havethe option of directly conjugating a PACPeD with a pendant silyl groupwith the steel of a stent manufactured as described in U.S. Pat. No.5,800,456, through the formation of a covalent bond. Alternatively, onecould copolymerize a PACPeD with pendant amine groups with apolyurethane, as in Example 16, and coat the stent with the polymer. Yetanother option would be to admix a PACPeD with polypropylene, as inExample 20, extrude the mixture into a stretchable film, and shrink wrapthe stent in the modified polymer film. As shown by this simple example,the diverse processes for the production of modified biomaterials usingnon-proteinaceous catalysts for the dismutation of superoxide allow thebio-engineer a wide variety of manufacturing techniques. A person ofordinary skill in the art of medical device design would be able todiscern which modified material, and which process of modification,would be best for the medical device being produced.

The biocompatible articles of the present invention may comprise severalbiomaterials modified with a non-proteinaceous catalyst for thedismutation of superoxide or a ligand precursor of a non-proteinaceouscatalyst for the dismutation of superoxide. This versatility will makethese materials especially useful in medical devices that are subject tocontinual wear and stress, such as joint replacement implants. Thepolyethylene “socket” polymer portion of the joint which allows alowered friction contact point in the implant could be injection moldedfrom a copolymer with the non-proteinaceous catalyst, while the metal“ball” portion of the joint which contacts the polyethylene could besurface covalently conjugated with a non-proteinaceous catalyst. Thus,an entire device with decreased inflammatory response may bemanufactured out of the modified biomaterials of the present invention,even though diverse materials are used in its construction. Another usefor the modified biomaterials, mentioned in the stent example above, iscoatings.

The chemical reactions described above are generally disclosed in termsof their broadest application to the preparation of the compounds ofthis invention. Occasionally, the reactions may not be applicable asdescribed to each compound included within the disclosed scope. Thecompounds for which this occurs will be readily recognized by thoseskilled in the art. In all such cases, either the reactions can besuccessfully performed by conventional modifications known to thoseskilled in the art, e.g., by appropriate protection of interferinggroups, by changing to alternative conventional reagents, by routinemodification of reaction conditions, and the like, or other reactionsdisclosed herein or otherwise conventional, will be applicable to thepreparation of the corresponding compounds of this invention. In allpreparative methods, all starting materials are known or readilyprepared from known starting materials.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and do not limit ofthe remainder of the disclosure in any way whatsoever.

EXAMPLES

All reagents were used as received without purification unless otherwiseindicated. All NMR spectra were obtained on a Varian VXR-300 or VXR-400nuclear magnetic resonance spectrometer. Qualitative and quantitativemass spectroscopy was run on a Finnigan MAT90, a Finnigan 4500 and aVG40-250T using m-nitrobenzyl alcohol (NBA) or m-nitrobenzylalcohol/LiCl (NBA+Li). Melting points (mp) are uncorrected.

Example 1 Preparation of Compounds Used in Template Synthesis

Chemicals, Solvents, and Materials.

UV Grade Acetonitrile (015-4) and Water (AH365-4) were obtained fromBurdick & Jackson (Muskegon, Mich.). Isopropanol (27,049-0),R,R-1,2-diaminocyclohexane (34,672-1), 2,6-diacetylpyridine (D880-1),2,6-pyridinedicarboxaldehyde (25,600-5), and trifluoroacetic acid(T6508) were purchased from Aldrich (Milwaukee, Wis.).2-(N-morpholino)-ethanesulfonic acid (475893) and its sodium salt(475894) were purchased from Calbiochem (La Jolla, Calif.).

N-(triphenylmethyl)-(1R,2R)-diaminocyclohexane:

To a solution of (1R,2R)-diaminocyclohexane (250 g, 2.19 mol) inanhydrous CH2Cl2 (3.5 L) at 0° C. was added, dropwise, a solution oftrityl chloride (254 g, 912 mol) in anhydrous CH2Cl2 (2 L) over 4 h. Theresulting mixture was allowed to warm to RT and stirred overnight. Thereaction mixture was washed with water until the pH of the aqueouswashes was below 8 (4×2 L) and dried over Na2SO4. Filtration andconcentration of the solvent afforded 322.5 g (99% yield) ofN-(triphenylmethyl)-(1R,2R)-diaminocyclohexane as a glass: 1H NMR (300MHz, DMSO-d6) d 7.50 (d, J=7.45 Hz, 6H), 7.26 (app t, J=7.45 Hz, 6H),7.16 (app t, J=7.25 Hz, 3H), 2.41 (dt, J=10.3, 2.62 Hz, 1H), 1.70 (m,1H), 1.54–0.60 (complex m, 8H). 13C NMR (75 MHz, DMSO-d6) dc 147.2 (s),128.4 (d), 127.3 (d), 69.9 (s), 59.0 (d), 54.4 (d), 36.6 (t), 32.5 (t),24.6 (t), 24.3 (t). MS (LRFAB) m/z=363 [M+Li]+.

Glyoxal bisimine of N-(triphenylmethyl)-(1R,2R)-diaminocyclohexane:

To a solution of N-(triphenylmethyl)-(1R,2R)-diaminocyclohexane (322.5g, 905 mmol) in methanol (4 L) was added glyoxal (51.9 ml of a 40%solution in water, 452.3 mmol), dropwise over 30 min. The resultingmixture was stirred for 16 h thereafter. The precipitated product wasisolated by filtration and dried in vacuo to afford 322.1 g (97% yield)of the bisimine product as a white solid: 1H NMR (300 MHz, CDCl3) d 7.87(s, 2H), 7.51 (d, J=8.1 Hz, 12H), 7.16–7.05 (m, 18H), 2.95 (b m, 2H),2.42 (b m, 2H), 1.98–0.81 (complex m, 18H). ). 13C NMR (100 MHz, CDCl3)161.67 (d), 147.24 (s), 147.22 (s), 128.90 (d), 128.81 (d), 127.73 (d),127.61 (d), 126.14 (d), 73.66 (s), 70.86 (d), 70.84 (d), 56.74 (d),32.45 (t), 31.77 (t), 24.02 (t), 23.62 (t). MS (LRES) m/z 757 [M+Na]+.

N,N′-Bis{(1R,2R)-[2-(Triphenylmethylamino)]cyclohexyl}-1,2-diaminoethane:

The glyoxal bisimine of N-(triphenylmethyl)-(1R,2R)-diaminocyclohexane(586 g, 798 mmol) was dissolved in THF (6 L) and treated with LiBH4(86.9 g, 4.00 mol) at RT. The mixture was stirred for 12 h at RT andtreated with a second 86.9 g (4.00 mol) portion of LiBH4. The reactionwas then warmed to 40° C. for 4 h thereafter. The reaction was carefullyquenched with water (1 L) and the THF was removed under reducedpressure. The residual slurry was partitioned between CH2Cl2 (3 L) andwater (1 additional L). The layers were separated and the aqueous layerwas extracted again with CH2Cl2 (1 L). The combined CH2Cl2 extracts weredried (MgSO4), filtered and concentrated to afford 590 g (˜100% crudeyield) ofN,N′-bis{(1R,2R)-[2-(triphenylmethylamino)]cyclohexyl}-1,2-diaminoethaneas a white foam: MS (LRES) m/z 739 [M+H]+.

N,N′-Bis{(1R,2R)-[2-(amino)]cyclohexyl}-1,2-diaminoethanetetrahydrochloride:

To a solution ofN,N′-bis{(1R,2R)-[2-(triphenylmethylamino)]cyclohexyl}-1,2-diaminoethane(590 g, 798 mmol) in acetone (3 L) was added concentrated HCl (1.5 L).The reaction was stirred for 2 h and concentrated. The residue waspartitioned between water (2 L) and CH2Cl2 (1 L). The layers wereseparated and the aqueous layer was concentrated and dried in vacuo toafford 257 g (80% yield) of the tetrahydrochloride salt as a granularoff-white solid: 1H NMR (300 MHz, CDCl3) 3.82–3.57 (complex m, 8H), 2.42(d, J=9.9 Hz, 2H), 2.29 (d, J=9.3 Hz, 2H), 2.02–1.86 (complex m, 4H),1.79–1.60 (complex m, 4H), 1.58–1.42 (complex m, 4H). 13C NMR (75 MHz,CDCl3) 59.1 (d), 51.3 (d), 40.8 (t), 29.2 (t), 26.0 (t), 22.3 (t), 22.2(t). MS (LRFAB) m/z 255 [M+H]+. The tetrahydrochoride salt can berecrystallized or precipitated from a viscous aqueous solution by theaddition of ethanol. This treatment removed all color.

Example 2 Template Synthesis of Compound 38

[Manganese (II)dichloro{(4R,9R,14R,19R)-3,10,13,20,26-pentaazatetracyclo[20.3.1.04,9.014,19]hexacosa-1(26),22(23),24-triene}].

In a 5-L flask N,N′-Bis{(1R,2R)-[2-(amino)]cyclohexyl}-1,2-diaminoethane tetrahydrochloride,(93.5 g, 234 mmol), was suspended in ethanol (3 L), treated with solidKOH (59.6 g of 88% material, 934 mmol), and the resultant mixturestirred at RT for 1 h. MnCl2 (anhydrous, 29.4 g, 233.5 mmol) was thenadded in one portion and the reaction was stirred at RT for 15 min. Tothis suspension was added 2,6-pyridinedicarboxaldehyde (31.6 g, 233.5mmol) and the resulting mixture was refluxed overnight. After 16 h, thetemplate reaction was complete: MS (LRFAB) m/z 443 [M−Cl]+. Seeaccompanying HPLC analyses. This material was taken on to the next step“as is”. The reaction mixture containing the template product in ethanolwas cooled to RT and treated (cautiously under Argon flow) with 10%Pd(C) (˜100 g in portions over the next 3–4 days) and ammonium formate(˜200 g also in portions over the next 3–4 days). The reaction wasrefluxed for 4 days. HPLC and MS analysis at this time showed completereduction. The catalyst was filtered through celite and the filtrate wasconcentrated to afford ca. 110 g of crude material. Recrystallizationfrom water afforded 50.0 g of the product in crop one as a pale yellowfinely divided solid. Upon sitting a second crop (12.5 g) was isolated.MS (LRFAB) m/z 447 [M−Cl]+. After drying the combined crops overnight invacuo at 70° C., a yield of 60.1 g (54%) was obtained. Analysis calc'dfor C21H35Cl2N5Mn: C, 52.18; H, 7.30; N, 14.49; Cl, 14.67. Found: C,51.89; H, 7.35; N, 14.26; Cl, 14.55.

The Synthesis is Diagramed below:

Example 3 Template Synthesis of Compound 40

[Manganese(II)dichloro(4R,9R,11R,14R,19R)-3,10,13,20,26-pentaaza-(2R,21R)-dimethyltetracyclo[20.3.1.0^(4,9).0^(14,19)]hexacose-1(25),22(26),23-triene,

To a stirred solution ofN,N′-Bis{(1R,2R)-[2-(amino)]cyclohexyl}-1,2-diaminoethanetetrahydrochloride (4.00 g, 10.0 mmol) in absolute ethanol (100 mL) wasadded KOH (2.55 g of ˜88% material, 40.0 mmol) and the mixture wasstirred at RT for 30 min. under an Ar atmosphere. MnCl₂ (anhydrous, 1.26g, 10.0 mmol) was then added and the suspension stirred for anadditional 30 min. or until MnCl₂ dissolved. At this point,2,6-diacetylpyridine (1.63 g, 10.0 mmol) was added to the green mixtureand after 30 minutes heating commenced. After refluxing for 5 d, themixture was dark red-brown. Mass spectrometry and HPLC analyses showedthat the reaction had gone to ³95% completion to give the bisimineMn(II) complex (˜94% purity by HPLC): ESI-MS: m/z (relative intensity)471/473 (100/32) [M−Cl]⁺; only traces of diacetylpyridine (˜5% by HPLC)and unreacted tetraamine complex (MS) were detected. The suspension wasallowed to cool to RT, and was stirred overnight. The next day, thesuspension was filtered (largely KCl) and dried in vacuo at 70° C.overnight. This material may be further purified by extractive work-upas follows: 69 g of the crude bisimine were dissolved in 1.2 L ofdistilled water. The yellow-orange solution was extracted with CH₂Cl₂(4×500 mL) and then 210 g of NaCl were added (final solution is ˜15% w/vin NaCl). The resulting suspension was extracted with CH₂Cl₂ (4×500 mL).The combined extracts were pooled, dried over MgSO₄, filtered, and thesolvent removed under reduced pressure. Upon drying in vacuo at 70° C.overnight, the product was isolated as an amorphous orange solid (ca. 50g, 78% recovery) with a purity of ca. 98% by HPLC.

Transfer Hydrogenation with Ammonium Formate.

The purified bisimine (1.0 g, 1.97 mmol) was dissolved in 100 mL ofanhydrous MeOH and the flask flushed with nitrogen while 3% Pd/C (0.5 g,50% by weight) was added. The suspension was heated and 10 mL of a MeOHsolution containing ammonium formate (1 g, 16 mmol) were added. After 30and 60 min. of reflux, a second and third portion of formate were added(16 mmol each). The suspension was allowed to cool to RT after 2 h ofreflux (at this point the supernatant was nearly colorless), filteredthrough celite and the solvent removed under reduced pressure. Theresulting yellow-green semisolid was stirred with 50 mL of CH₂Cl₂ for5–10 min., filtered, and the solvent removed once more. The remainingyellow-green foam consisted of ˜95% S,S- and S,R-isomers in a 3.8:1ratio as determined by HPLC.The synthesis is diagramed below:

Purification Protocol.

Extraction of Comound 40 (S,S-isomer) from the Crude Mixture Obtainedfrom Transfer Hydrogenation.

Crude product isolated after transfer hydrogenation (9.3 g) wasdissolved in water (370 ml) and extracted with DCM (4×185 ml). Allorganic extracts and aqueous phase were analyzed by HPLC to follow theprogress of extraction. Analysis was performed either in a complex formor after release of the free ligand. Recovery of R,S- and S,S-isomerfrom DCM (1+4, extracts from water): (2.42 g+1.18 g+1.24 g=4.84 g).After 4 extraction with DCM no R,S-isomer was detected by HPLC inaqueous phase. Then solid NaCl was added (10.82 g) to make up 0.5 Msolution and S,S-complex (Compound 40) was extracted 4 times with DCM(370 ml each). Most of the S,S-isomer was extracted into the 1^(st) DCMextract (purity by HPLC>94%). Impurities (others than R,S-isomer) wereextracted at 4–6% level). After evaporaion of the first two DCM extractsand drying under high vacuum 3.04 g of S,S-isomer Compound 40 wasobtained with purity 94%. The product was further purified by HPLC usingYMC C18 column or by flash chromatography over C18 silica gel column.

Purification by Preparative HPLC

Compound 40 (200 mg) obtained from extraction (purity 91%) was dissolvedin water (1.0 ml) and applied onto YMC CombiPrep column (20 mm×50 mm,ODS AQ 5 um 120A). The product was eluted using gradient—B 10 to 50% in10 min, where A: 0.5M NaCl and B: Acetonitrile-Water (4:1), flow rate 25ml/min, detection at 1=265 nm. Fractions with purity >99% (8 to 20, each5 ml) were combined and the solvents were evaporated to dryness. Theresidue was partitioned between 6 ml water and 10 ml of DCM. Seperatedlayers, extracted aqueous layer with 3×10 ml DCM. Combined DCM layers,dried over Na₂SO₄, filtered and evaporated solvents to off-white foam,Obtained 97 mg, 48%. ESMS m/z 475 [M−Cl]⁺ Calcd for C₂₃H₃₉Cl₂N₅Mn.

Purification of Compound 40 by Flash Chromatography Over C18 Silica Gel

40 g of Bakerbond Octadecyl C₁₈ packing was packed into a 25 mm×130 mmcolumn. Column was equilibrated with CH₃CN (300 ml), 1:1=H₂0:CH₃CN (200ml), 15% CH₃CN in H₂O (200 ml) and 15% CH₃CN in 0.5 M NaCl (200 ml).Compound 40 (1 g) obtained from extraction (purity 94% by HPLC) wasdissolved in 3 ml of H₂O and applied onto the column. The product waseluted with 15% CH3CN in 0.5M NaCl. Fractions were analyzed by HPLC.HPLC conditions were as follows: YMC C₁₈ column, 3 ml/min, l=265 nm,B=10–50% in 9 min, where A=0.5 M NaCl in H₂0 and B=CH₃CN:H₂0=4:1. TheS,S-isomer eluted in fractions 51–170. Fractions with purity >95%(80–170) were combined and the solution was concentrated to 80 ml andextracted 2× with DCM. (40 ml each). Obtained 0.64 g (yield 64%) of theS,S-isomer (Compound 40), 100% pure by HPLC ESMS m/z 475 [M−Cl]⁺ Calcdfor C₂₃H₃₉Cl₂N₅Mn.

Example 4 Template Synthesis of Compound 42

Synthesis of 4-chloro-2,6-pyridinedicarboxaldehyde.

4-Chloro-2,6-dicarbomethoxypyridine:

Anhydrous chelidamic acid (230 g, 1.14 mol) was partially dissolved inCHCl3 (2 L) while stirring under N2. Then, over a period of 3 h, PCl5(1,000 g, 4.8 mol) was added as a solid to the cream-colored suspension.Considerable gas evolution occurred with each solid addition. After 17h, the white mixture was heated to reflux and a light yellow solutionresulted within an hour. Seven hours later, heating was discontinued.The light suspension was treated with MeOH (1.25 L), added dropwise over6.5 h. Then, after gas evolution had ceased, the solution wasconcentrated under reduced pressure and the off-white slurry that formedadded to deionized water and vacuum-filtered. The residue was washedwith more water (˜5 L) until the pH of the filtrate was neutral. Theresidue was dried overnight in vacuo at 50–60° C. to afford4-chloro-2,6-dicarbomethoxypyridine as white needles (175 g, 66%); m.p.132–134 ° C. 1H-NMR is consistent with the structure.

4-Chloro-2,6-pyridinedimethanol:

The methyl ester prepared as above (675 g, 2.94 mmol) was partiallydissolved in MeOH (16 L) and stirred under N2 with cooling in an icebath. NaBH4 (500 g, 13.2 mol) was added as a solid in portions over thenext 20 h. Over the course of 48 h, the reaction went from orange to redto yellow-green. Then, the temperature was allowed to reach RTovernight. After this period, the mixture was refluxed for 16 h, thencooled over 6 h to afford a clear yellow-green solution. Acetone (3.1 L)was added over 1.5 h, then the yellow solution was refluxed for 2 h.Concentration under reduced pressure yielded an amorphous light yellowgum. The gum was taken up in saturated Na2CO3 and heated to ˜80 ° C. for1 h. Upon cooling overnight, the viscous yellow supernatant wasseparated from the white precipitate by vacuum filtration. The solid waswashed with CHCl3 (350 mL), then taken in THF (4.5 L) and refluxed for30 min., then filtered. The filtrate was concentrated under removedpressure, the solid residue washed with CHCl3, then dried in vacuoovernight to afford the diol product (375 g, 68%) as a white solid.1H-NMR is consistent with the structure.

4-Chloro-2,6-pyridinedicarboxaldehyde:

A solution of oxalyl chloride (110 mL, 1.27 mol) in CH2Cl2 (575 mL) wascooled to −60 ° C. and stirred under N2. To this solution was added asolution of dimethylsulfoxide (238 mL, 3.35 mol) in CH2Cl2 (575 mL) viacannula. Addition proceeded with vigorous gas evolution and a mildexothermic reaction over 1.5 h. After stirring for 10 min. a solution ofthe diol (100 g, 0.58 mol) in DMSO (288 mL) was added via cannula over aperiod of 30 min. The previously yellow solution turned into asuspension. After 2 h at −60 ° C., Et3N (1.5 L) was added dropwise over1 h. After addition was complete and 30 min. had passed, the mixture waspoured over water (2 L), shaken and allowed to settle. The organic layerwas separated and the aqueous layer extracted with CH2Cl2 (4×300 mL).The combined CH2Cl2 layers were dried over MgSO4 and concentrated underreduced pressure to afford a combination of gray-yellow solid and areddish liquid. The dark yellow solid was collected by filtration usingEt2O to rinse out. This material was dissolved in 1L of CH2Cl2 andpassed through a bed of SiO2 (˜800 cm3) eluting with more CH2Cl2. Atotal of 65 g (67%) of the dialdehyde product were collected in thisfashion. 1H-NMR is consistent with the structure.

Preparation of 4-chlorobisimine by Template Cyclization.

Bis-R,R-Cyclohexane tetraamine.4HCl (2.57 g, 6.42 mmol) was suspended inabsolute EtOH (64 mL) and stirred under Ar. Pellets of KOH (1.65 g of87.4% material, 25.68 mmol) were added and the suspension stirred for 30min. until the pellets dissolved. After this period, MnCl2 (anhydrous,0.806 g, 6.42 mmol) was added and allowed to stir for 1–2 h until thesuspension turned greenish and all the MnCl2 dissolved.4-Chloro-2,6-pyridinedicarboxaldehyde (1.09 g, 6.42 mmol) was added as asolid and the mixture stirred at room temperature for 30 min., thenheated to reflux. The suspension gradually turns red-orange and after 48hours it was cooled to room temperature. The mixture was filteredthrough a 10 m pore-size funnel and the solvent removed under reducedpressure to yield the desired product (3.47 g, 105%, contains someinorganic salts) as a red-orange solid.

NaBH4 Reduction.

The bis-imine complex (1.89 g, 3.68 mmol) was dissolved in anhydrousMeOH (50 mL) and stirred under Ar in an ice-water bath. Solid NaBH4(0.278 g, 7.36 mmol) was added in one portion resulting in gasevolution. After 30 min., an additional portion of NaBH4 (7.36 mmol) wasadded and the mixture allowed to warm to RT, and stirred overnight. Athird portion of NaBH4 (7.36 mmol) was added at 0EC, then the mixtureallowed to warm and stirred overnight. After this period, MS stillshowed starting material remaining. A fourth, fifth, and sixth portionof NaBH4 (7.36 mmol each) were added with 2 hours passing in betweenaddition.

After 24 h at RT, the lightly colored solution was carefully added onto100 mL of saturated NaCl solution, and MeOH removed under reducedpressure. CH2Cl2 (100 mL) was added and the aqueous layer extracted(2×). The organic layer s were combined, dried over MgSO4, filtered andthe solvent removed to afford, upon drying in vacuo, 2.1 g of crudematerial (60% product by HPLC). This material was purified by SiO2 flashchromatography using 1 à 3% MeOH:CH2Cl2 as eluent. Selected fractionsyielded 0.77 g (40%) of HPLC-homogeneous material. ESI-MS: m/z (relativeintensity) 481/479 (100/32) [M−Cl]+; and 223/221 (100/32) [M−2Cl]2+.The Synthesis is Diagramed below:

Example 5 Synthesis of Compound 43 from Compound 42

To a solution of 1.2% (w/v) 2-mercaptoethylamine (1 eq) in ethanol at 0°C. was added sodium ethoxide (1.1 eq) to generate the thiolate. Afterstirring for 1.75 h, the thiolate solution was added dropwise to asolution of 1.3% (w/v) SC 74897 (1 eq) in DMF at 0° C. The reactionmixture was allowed to stir overnight. The solvent was removed in vacuo,the product mixture was extracted with methylene chloride, andconcentrated down in vacuo. Flash column chromatography using methylenechloride:methanol (9:1) as the eluent was used for purification, whichwas monitored via HPLC.

The synthesis is illustrated below:

Example 6 Catalytic Hydrogenation of the Bisimine

Transfer Hydrogenation with Ammonium Formate.

The purified bisimine (1.0 g, 1.97 mmol) was dissolved in 100 mL ofanhydrous MeOH and the flask flushed with nitrogen while 3% Pd/C (0.5 g,50% by weight) was added. The suspension was heated and 10 mL of a MeOHsolution containing ammonium formate (1 g, 16 mmol) were added. After 30and 60 min. of reflux, a second and third portion of formate were added(16 mmol each). The suspension was allowed to cool to RT after 2 h ofreflux (at this point the supernatant was nearly colorless), filteredthrough celite and the solvent removed under reduced pressure. Theresulting yellow-green semisolid was stirred with 50 mL of CH₂Cl₂ for5–10 min., filtered, and the solvent removed once more. The remainingyellow-green foam consisted of ˜95% S,S- and S,R-isomers in a 3.8:1ratio as determined by HPLC.

% Area by HPLC^(d) Concentration Catalyst % by Time Free SS- SR-(nM)^(a) (% Pd.C)^(b) Weight (hours) Ligand Monoimine isomer isomerRatio 20 10 50 2 — — 68 32 2.13 20 5 50 2 2 — 75 23 3.26 20 5 10 4 2 764 27 2.37 20 3 50 2 2 2 75 21 3.57 50 3 50 2 4 — 70 25 2.80 100 3 50 29 <1 64 26 2.46 ^(a)Solvent is anhydrous MeOH. ^(b)Available fromAldrich. ^(c)Reflux time. ^(d)Conditions: 3 mL/min. 10–50% B over 9 min.is (8:2 v/v) MeCN: water, A is 0.5N aq. NaCl. UV-detection at 265 nm.

Example 7 Conjugation of Polyethylene Terephthalate with a PACPeDCatalyst

A. Denier Reduction (Alkaline Hydrolysis) of Poly(ethyleneTerephthalate) (PET) Film

20 mm×50 mm×5 mm PET film (37% crystallinity) pieces were cleaned bymixing for 30 min in a 1% (w/w) aqueous Na₂CO₃ solution (250 mL) at 75°C. The film pieces were removed and washed 30 min in water (HPLC grade,250 mL) at 75° C. The pieces were next hydrolyzed for 30 min in a 0.5%(w/w) aqueous NaOH solution (250 mL) at 100° C. The film pieces added toa 1.2% (w/w) aqueous conc. HCl solution (250 mL) at room temperature.Finally, the film pieces were thoroughly rinsed in a stream of water(HPLC grade) at room temperature and dried to constant weight in vacuo.

B. Preparation of the Acid Chloride

A magnetic stir bar and anhydrous acetonitrile (50 mL) were added to adry 100 mL round bottom flask. To the stirring solvent was added onepiece of hydrolyzed film, pyridine (0.078 g, 9.89×10⁻⁴ mol), and thionylchloride (0.167 g, 1.4×10⁻³ mol). After stirring for 24 h at roomtemperature, the film was removed and thoroughly rinsed in freshacetonitrile. After drying to constant weight in vacuo, elementalanalysis showed the presence of chlorine in the film.

C. Reaction with Amino Functional PACPeD

A magnetic stir bar and anhydrous acetonitrile (50 mL) were added to adry 100 mL round bottom flask. Amino functional Compound 43 (0.138 g,1.86×10⁻⁴ mol) was added. Once in solution, the film step Bwas added andthe reaction mixture was heated to reflux. After 24 h at reflux, thefilm was removed and rinsed in fresh acetonitrile before drying toconstant weight in vacuo. ICAP analysis of the film revealed thepresence of manganese.

The conjugation scheme is illustrated by the following:

Example 8 Conjugation of Acrylic Acid Modified Polyethylene with aPACPeD Catalyst

A. Grafting of Acrylic Acid to PET Films

Pieces of 20 mm×50 mm×5 mm PET film (37% crystallinity) were usedwithout cleaning. Film pieces were swollen in 80° C. 1,2-dichloroethanefor 1 h. The films were then dried to constant weight in vacuo.

Swollen film pieces were added to a 0.08 M benzoyl peroxide in anhydroustoluene solution (125 mL). After mixing for 1 h at room temperature, thefilm pieces were removed, rinsed in fresh anhydrous toluene, and driedto constant weight in vacuo.

Next, the films were immersed in a 30 mL vial containing a 2 M acrylicacid (freshly distilled) and 0.1 mM Mohr's salt {(NH₄)₂Fe(SO₄)₂x 6 H₂O}aqueous solution (25 mL). The vial was purged with nitrogen, sealed, andimmersed in an 80° C. oil bath. The film pieces were stirred for 20–24 hat 80° C. before removal and rinsing for several minutes in hot runningtap water followed by a stream of room temperature water (HPLC grade).After drying overnight in vacuo, the acrylic acid grafted films wereimmersed for 5 h in boiling water (HPLC grade) and dried to constantweight in vacuo.

Preparation of the Hydrolyzed PET Film and Conjugation with the PACPeDCatalyst Proceeded as Described in Example 7.

The Conjugation Scheme is Illustrated by the Following:

Example 9 Surface Covalent Conjugation of Compound 43 withPoly(etherurethaneurea)

The poly(etherurethaneurea) (PEUU) (M_(n)=50,000) used for conjugationwas a segmented block copolymer consisting of methylene di(p-phenylisocyanate) (MDI), ethylene diamine, and poly(tetramethyleneglycol)(PTMG, M_(n)=2000). The ethylene diamine chain extended MDI makes up thehard segment and the PTMG makes up the soft segment. PEUU films weresolvent cast from a solution of 20% PEUU in N,N-dimethylacetamide (DMAC)and allowed to dry under nitrogen for ˜2 days. Films were further driedin vacuo before being cut into ˜5 mm diameter disks of ˜0.3 mmthickness.

PEUU disks were functionalized in a solution of 5.4% (w/v) HMDI inanhydrous toluene with triethyl amine added to serve as the catalyst.The reaction was allowed to stir at 55–60° C. for 24 h, the disks werethoroughly washed with anhydrous toluene, and dried. Disks were added toa solution of 0.3% (w/v) Compound 43 in anhydrous toluene and allowed tostir at 55–60° C. for 24 h. The disks were washed with toluene,methanol, and water to remove any unbound SOD mimic prior toimplantation. By inductively coupled argon plasma analysis (ICAP,Galbraith Laboratories, Knoxville, Tenn.) of manganese there was 3.0%catalyst by weight.

To obtain a lower concentration of Compound 43, a solution of 0.7% (w/v)HMDI in anhydrous toluene (15 h) and a solution of 0.1% (w/v) Compound43 in anhydrous toluene (24 h) was used. ICAP analysis of manganeseindicated 0.6% Compound 43 by weight.

Example 10

Njugation of Compound 43 and Poly(ethylene Acrylic Acid)

UHMWPE was melt blended with poly(ethylene-co-acrylic acid) in a ratioof 7:3 in a DACA twin screw at 175° C. Blends were cryoground and meltpressed into films with 5000 psi at 175° C. for 10 minutes. Films werecut into 5 mm diameter disks of ˜0.5 mm thickness.

PE disks were chlorinated in a solution of 0.2% (w/v) thionyl chloridein acetonitrile. Pyridine was added to scavenge the HCl formed. Themixture was allowed to stir overnight, the disks were filtered, washedthoroughly with acetonitrile, and dried. Chlorinated disks were added toa solution of 0.1% (w/v) Compound 43 in acetonitrile, heated to refluxfor 4 hours, and allowed to react at room temperature overnight. Thedisks were filtered and washed with acetonitrile and water. ICAPanalysis for manganese indicated 1% Compound 43 by weight.

To obtain a lower concentration of Compound 43, the chlorinated diskswere added to a solution of 0.02% (w/v) Compound 43 in DMSO and heatedat 60° C. overnight. The disks were filtered and washed repeatedly withmethanol and water. ICAP analysis for manganese indicated 0.06% Compound43 by weight.

The synthesis is diagramed below:

Example 11 Surface Covalent Conjugation of Compound 52 withPoly(ethylene-co-acrylic Acid) via a PEO Linker

To a flask under a N₂ purge was added polyethyene-co-polyacrylic acid(0.4 g) (15% acrylic acid by weight), DMSO (100 mL), and EDC (0.3192 g).The mixture was allowed to stir for 1 h, then polyoxyethyelene bisamine(amino-PEO) (2.65 g) (Sigma, MW=3400) was added. The mixture was allowedto stir overnight. The mixture was precipitated with water and dried invacuo yielding 0.37 g of white powder. The powder was 1.9% N by weightas determined by elemental analysis.

To a flask under a N₂ purge was added EDC (0.0112 g), Compound 52 (0.031g), and CH₂Cl₂. The solution was allowed to stir for 2 h at roomtemperature and then the amino terminated PEO functionalizedpolyethyene-co-polyacrylic acid (0.2 g) was added and the solution wasallowed to stir overnight. Methanol (50 mL) was added to the solution,the precipitate was filtered off, washed with methanol and water, anddried in vacuo overnight. By ICAP analysis, 0.26% of manganese by weightwas present.The Synthesis is Diagramed below:

Example 12 Surface Covalent Conjugation of Compound 43 withPoly(etherurethane Urea) Coated Tantalum

To a 0.5% (w/v) PEMP solution in DMAC was added 3-isocyanatopropyltriethoxysilane (3% w/v) and triethyl amine. The reaction mixture washeated to 55–60° C. for 18 h and then precipitated with ethanol,filtered, and dried. A solution of 1% (w/v) polymer in DMAc was formed.To the oxidized tantalum disks was added polymer solution and water(50:1, v:v). After agitation for 24 h, the disks were cured at 110° C.for 1 h, rinsed with DMAc, and dried. Half of the disks were set asidefor use as controls during implantation. To the PEUU coated disks wasadded a solution of 5% (w/v) HMDI in anhydrous toluene and allowed toreact at 55–60° C. for 24 h. After washing with anhydrous toluene anddrying, a solution of 1% (w/v) Compound 43 in 1,1-dichloroethane wasadded and allowed to react for 24 h at 55–60° C. The disks were thenwashed with 1,1-dichloroethane, methanol, and water. After drying, ESCAwas obtained and indicated a 1.2% atomic fraction of manganese on thesurface.The Synthesis is Diagramed below:

Example 13 Surface Covalent Conjugation of Compound 43 with Tantalum

Disks with a diameter of 6 mm were punched out from 0.25 mm thicktantalum sheets and the edges smoothed.

Tantalum disks were initially oxidized using a H₂SO₄:30% H₂O₂ (1:1, v:v)solution. 3-isocyanatopropyl triethoxysilane (2% w/v) was added to anethanol-water solution (0.8% water by weight) of pH=5 (adjusted withacetic acid) and agitated for 5 min. To the oxidized tantalum disks wasadded the silane and after agitation for 10 min. the disks were quicklyrinsed with ethanol, and cured at 110° C. for 1 h. Half of the diskswere set aside for use as controls during implantation. To thepolysiloxane layered disks was added a solution of 0.5% (w/v). Compound43 in DMAc and allowed to react at 60–65° C. for 24 h. After washingwith DMAC and drying, one disk was studied by electron scanning forchemical analysis (ESCA), which indicated a 0.5% atomic fraction ofmanganese on the surface.

The synthesis is diagramed below:

Example 14 Surface Covalent Conjugation of Compound 43 with Collagen

To a flask 0.5 g of bovine collagen (insoluble, type I from Achillestendon) was suspended in a 4% solution of 1,4 butanediol diglycidylether in a buffer solution. The solution was stirred overnight. Thesolution was then centrifuged for about 10 minutes,and the supernatentwas decanted. Any residual, adsorbed diglycidyl ether was removed fromthe above partially cross-linked collagen by repeated washings withmethanol. At this point, the washed collagen was immersed in a solutionof Compound 43 (100 mg in 50 ml) of the same buffer used in the reactionset-forth above. The contents were stirred at ambient temperature in around bottomed flask overnight. At the end of this period, the contentswere centrifuged and washed as in the earlier step to remove anyunreacted Compound 43. The recovered collagen (0.304 g) was driedovernight in a vacuum oven at a temperature of 50° C.

ICAP analysis indicated 0.18% Mn in the collagen corresponding to 1.83%binding of Compound 43.

Example 15 Surface Covalent Conjugation of Compound 43 with HyaluronicAcid

To a solution of 0.05 g of sodium salt of hyaluronic acid (Sigma H53388,Mol.Wt., 1.3×10⁶) in 16.7 ml distilled water was added 0.070 g ofCompound 43 and the pH of the solution lowered from 9.3 to 6.8 bycareful addition of 0.1M HCl. A solution of1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,[EDC.HCl](0.012 g) and 1-Hydroxy-7-azabenzotriazole [HOAT] (0.009 g) indimethyl-sulfoxide(DMSO)-water (0.5 ml; 1:1, v/v) was added and the pHwas adjusted from 5.2 to 6.8 and maintained at 6.8 by incrementaladditions of 0.1 M sodium hydroxide. The contents were stirred overnightat ambient temperature. After 20 hr, the pH was readjusted to 6.8 from6.94 and again stirred overnight for a total of 48 hr. At the end ofthis period, pH of the solution was again adjusted to 7.0 and dialyzedin Pierce Slide-a-dialyzer cassettes (Mol.Wt. cuttoff:10,000) againstdistilled water for 65 hr. Dialyzed contents from the cassettes weresyringed out (16.7 ml) and 0.8 g of NaCl was added to obtain a 5% saltsolution. The reaction product was precipitated by the addition ofethanol (×3 to 48 ml). The cotton-like white solid was recovered byfiltration, dried under vacuum overnight. A total of 0.0523 g of theisolated product on ICAP analysis showed a 0.21% Mn corresponding to2.1% binding of Compound 43 to hyaluronic acid.The Synthesis is Diagramed below:

Example 16 Copolymerization of Compound 16 with Polyureaurethane

A solution of vacuum distilled 4,4′-methylenebis(phenylene isocyanate)(MDI) is prepared in N,N′-dimethylacetamide (DMA). Polytetramethyleneoxide (PTMO), dehydrated under vacuum at 45–50^(E)C for 24 h andstannous octoate catalyst are subsequently added to the stirred MDIsolution at room temperature. The concentration of the reactants insolution is about 15% w/v and of the catalyst is 0.4–0.5% by weight ofthe reactants. After reacting at 60–65EC for 1 h, the mixture is cooledto 30EC. Ethylene diamine (ED) and diamino Compound 16 are then addedand the temperature gradually brought back to 60–65^(E)C. This is toprevent an excessively rapid reaction of the highly reactive aliphaticamine groups with isocyanates. The reaction is continued for anadditional hour at about 65EC. The entire synthesis is carried out undera continuous purge of dry nitrogen. Molar ratios of MDI, ED, SODm, andPTMO and the molecular weight of PTMO are varied to producepolureaurethanes of varying hardness. The polymers are precipitated in asuitable non-solvent like methanol and dried in a vacuum oven at 70–75ECfor about a week. Films for physical testing and implantation in ratsare prepared by a conventional spin-casting technique followed by vacuumdrying at 70EC for 4 days.The Polymer Produced by this Method is Represented Diagrammaticallybelow:

Example 17 Copolymerization of Compound 53 with Methacrylic

Synthesis of Methacryl Functional SODm:

A ˜10 percent (w/v) solution of hydroxy (or amino) functional PACPeD in1,2-dichloroethane is placed in a three necked flask equipped with astirrer, a dropping funnel and a reflux condenser. To this solution, a˜10 percent (w/v) solution of methacryloyl chloride in 1,2dichloroethane is added dropwise at 0□C followed by pyridine. Themixture is stirred at room temperature for about 16 h. The reactionmixture is filtered to remove pyridine hydrochloride and the filtrate isconcentrated under reduced pressure. The residue is dissolved inmethanol and the methacryl functional SODm is recovered by columnchromatography.

Synthesis of (Meth)acrylic Copolymers Containing SODm:

Mixtures of freshly distilled methyl methacrylate and Compound 53 aredissolved in toluene (˜10%w/v) and transferred to a three necked flaskequipped with a stirrer, a nitrogen inlet/outlet and a reflux condenser.Azodiisobutyronitrile (1% on the weight of monomer mixture) is added andthe solution is purged free of occluded air by oxygen free nitrogen. Thecontents are heated to 50□C and maintained at that temperature stirredunder a nitrogen sweep for 48 h. The polymer solution is then slowlypoured with good stirring into a large excess of methanol to recover thecopolymer. The recovered copolymer can be further purified byreprecipitation from a toluene solution in methanol.

This Synthesis Results in the Following Polymer:

Example 18 Copolymerization of Hexamethylene Diamine with Compound 16

Synthesis of Poly(hexamethylene -co-SODm Sebacamide):

A mixture of hexamethylene diamine (HMD) and diamino Compound 16 isdissolved in absolute ethanol and added to a solution of sebacic acid inabsolute ethanol. The mixing is accompanied by spontaneous warming.Crystallization soon occurs. After standing overnight, the salt isfiltered, washed with cold absolute ethanol and air dried to constantweight. About 2% excess of HMD is used to promote a salt rich indiamine. HMD being the more volatile component, is lost during saltdrying or during polycondensation.

The dried salt is heated in a suitable reactor with good stirring firstto 2150EC for about an hour and then to 2700EC. After 30–60 minuteheating under atomospheric pressure, the heating is continued undervacuum for about an hour. The polymer is then cooled under nitrogen andrecovered.

Example 19 Copolymerization of Compound 27 with Tetramethylene Glycoland Isophthalate

A three necked flask equipped with a nitrogen inlet tube extending belowthe surface of the reaction mixture, a mechanical stirrer, and an exittube for nitrogen and evolved hydrogen chloride is flushed with nitrogenand charged first with a isophthaloyl chloride followed by astoichiometric amount of a mixture of tetramethylene glycol and Compound27 ligand. The heat of reaction would cause the isophthaloyl chloride tomelt. The reaction is stirred vigorously and nitrogen is passed throughthe reaction mixture to drive away the hydrogen chloride (and collectedin an external trap). The temperature of the reaction is then raised to180° C. and held at that temperature for 1 hour. During the last 10minutes of the 180° C. heating cycle, the last of the hydrogen chlorideis removed by reducing the pressure to 0.5–1.0 mm. The copolymer isobtained as a white solid. Compound 27 in the polymer backbone is thencomplexed with manganese chloride.

Example 20 Admixture of Compound 38 with Polypropylene

Compound 38 was determined to be thermally stable up to 350EC. 0.105 gof Compound 38 was added to 4.9 g of cryoground polypropylene. Themixture was melted at 250EC and extruded into a strand and a fiber. Inthis manner, a polypropylene modified with a non-proteinaceous catalyst,2% by weight, was made. The product strand was cryoground and extractedwith pure water. Active Compound 38, as confirmed by both stopped-flowkinetic analysis and HPLC-UV spectroscopy, was extracted from thestrand. The concentration of Compound 38 in the water was has beencalculated to correspond to approximately a 10% elution of the admixedCompound 38 from the cryoground polymer. This suggests that thepolypropylene would release active PACPeD catalyst at the plastic-humanbody tissue interface where it would serve to reduce inflammation. Otherpolymers which melt under 300EC and which would be suitable for use inthe above process (with any appropriate temperature changes) arepolyethylene, polyethylene terephthalate, and polyamides.

Example 21 In Vivo Evaluation of the Inflammatory Response to SeveralSurface Covalently Conjugated Polymers and Metal

Samples of biomaterials, with and without PACPeD catalysts, in the formof 5–6 mm discs were implanted subcutaneously on the dorsal surface offemale, 250–300 gm, Sprague Dawley rats. All disks were sterilized bythree brief rinses in 70% alcohol followed by five brief rinses insterile saline (0.9% NaCl) just prior to implantation. All biomaterialswere conjugated with Compound 43. Polyurethane implants were bathed insterile saline for one hour prior to sterilization in ethanol andimplantation. Animals were initially anesthetized with 5% oxygen and 95%carbon dioxide to shave the dorsal region followed by methefane vaporadministered through a nose cone during surgery. Following a sterilescrub of the surgical field, a 5 to 6 cm incision through the skin wasmade along the dorsal midline, a pocket in the interstitial fascia wasprepared with a blunt scissors and the implant disks were inserted. Thewound was closed with surgical staples. All animals were ambulatorywithin one hour of anesthesia. For the polyurethane and polyethylenestudy, each animal received an untreated control and two PACPeD treateddisks at a high and low dose. For the tantalum study, each animalreceived a total of four disks, two controls containing to two types oflinkers and two matched PACPeD treated discs. After periods of 3, 7, 14,and 28 days, animals were sacrificed with 100% carbon dioxide and thedorsal skin flap was removed and fixed in 10% neutral buffered formalin.The skin tissue was pinned upside down for photography of the implantsin situ and the individual implants with surrounding tissue were excisedand processed in paraffin for light microscopy. PE and PEUU implantswere sectioned with the implants embedded in the paraffin block.Tantalum implants were embedded in paraffin and the paraffin block wascut in half with a low speed diamond saw. These halves were then cooledin liquid nitrogen and fractured with a cold razor blade to expose thetantalum disc. The disc was then removed from the block leaving theimplant capsule intact. The tissue blocks were remelted and mounted toexpose the implant capsule for microtomy. Sections were stained withhematoxylin and eosin and Gomori trichrome (Sigma, St. Louis Mo.). Inaddition, sections were stained immunohistochemically to identifymonocyte-derived macrophages with a macrophage specific antibody, ED1(Chemicon Inc., Temecula, Calif.). The cellular composition of theimplant capsule and surrounding tissue and the matrix composition werescored visually. Measurements of foreign body giant cells number andcapsule thickness were made by visual inspection and by computer basedmeasurement of digital micrographs. All data were reported as the meanand standard deviation.

Results

Conjugated Polyethylene

Histological analysis was performed on triplicate sets of untreatedcontrol PE disks and two PACPeD treated PE disks having with either alow (0.06%) or high (1.1% (w/w)) level of PACPeD after 3, 7, 14 and 28days of implantation. These times were selected in order to observe theacute inflammation phase and the progression to a chronic inflammation.Although differences in the healing response were observed at each time,major differences were apparent at 3 and 28 days. At 3 days, control PEdisks were completely surrounded by a dense granulation tissueconsisting of neutrophils and macrophages, FIG. 5A. Small blood vesselsin tissue adjacent to the implant contained many adherent monocytes andleukocytes and some in various stages of transendothelial migration fromblood to implant tissue. In striking contrast, the granulation tissuesurrounding low and high dose PACPeD-PE, FIGS. 5B and 5C, contained veryfew and no neutrophils, respectively. Numerous macrophages were presenton the low dose implant and labeled with ED1 antibody to suggest thatthey are monocyte derived. In the high dose implant capsule, the numberof macrophages was greatly reduced and fibroblast like cells constitutedthe major cell type. In addition, blood vessels adjacent to thePACPeD-PE implants contained no adherent leukocytes or monocytes.

After 28 days equally remarkable differences were observed. In thecontrol, foreign body giant cells (FBGCs) formed a layer between theimplant and the implant capsule tissue, FIG. 6A, to indicate thatchronic inflammation was underway. FBGCs also filled the many scratchesthat formed the rough PE surface. The implant capsule tissue consistedof layered fibroblasts, some ED1 positive macrophages, a few neutrophilsand collagen matrix. For the low level PACPeD disks, the capsule had amarked reduction in FBGCs on the surface and in number of cells in thecapsule in comparison to control, FIG. 6B. With the high PACPeD-PEdisks, FBGCs were rarely observed, FIG. 6C. The number of FBGCs observedin two independent sections per disk for a total of six counts pertreatment group were averaged and revealed a statistically significantdecrease in FBGC with PACPeD-PE over control, FIG. 7. In addition, thethickness of the implant capsule as measured from the same sections wassignificantly reduced in comparison to untreated control PE.

Polyurethane

Histological analysis was performed on triplicate sets of untreatedcontrol PEUU disks and two PACPeD treated PE disks having with either alow (0.6%) or high (3.0% w/w) level of PACPeD after 3, 7, 14 and 28 daysof implantation. Although, PEUU is well known to be less inflammatorythan polyethylene, the effect of surface bound PACPeD mimic was obviousand similar to that observed for PE disks at 3 and 28 days. At 3 days,implant capsules of control PEUU disks contained neutrophils and ED1positive macrophages although their numbers were estimated to be twoorders of magnitude less than PE control. Capsules surrounding the lowlevel PACPeD-PEUU implants had a markedly reduced but detectable numberof neutrophils with macrophages and fibroblast being predominant. As wasobserved for the PACPeD-PE implants, capsule tissue around the high dosePACPeD-PEUU disks contained no observable neutrophils and a reducednumber of macrophages.

At 28 days, implant capsules around the PEUU control disks had a layerof adherent FBGCs and layers of fibroblasts, ED1 positive macrophagesand collagen matrix, FIG. 8A. With the low level PACPeD, FIG. 8B, thenumber of FBGCs was reduced although the implant capsule containedfibroblast and fewer ED1 positive macrophages and had a thicknesssimilar to the control. The high level PACPeD-PEUU disk capsule had veryfew FBGCs and the capsule thickness was estimated to be one half of thecontrol capsule, FIG. 8C.

It is well known that PEUU is susceptible to biodegradation in vivoleading to the formation of surface pits and cracks. To monitor thiseffect in control and functionalized disks, scanning electronmicroscopy, SEM, was used to examine non-implanted disks and 28 dayimplanted disks, FIGS. 1–3. The non-implanted PEUU film showed a smoothsurface with no cracks or pitted areas, FIG. 1. The implanted controlPEUU sample after 28 days contained large, multiple cracks and areaswhere the surface had been eroded, FIG. 2. The implanted PACPeD-PEUUsample showed no obvious differences compared to the non-implantedcontrol, FIG. 3. Hence, in addition to inhibiting both acute and chronicinflammatory responses, PACPeD linked to PEUU surface inhibited surfacedegradation observed at 28 days.

Tantalum

Tantalum disks treated with either the silane linker or the PACPeD andsilane linker were implanted for 3 and 28 days. The healing response wassimilar to that seen for treated and untreated polymers. After 3 days, aneutrophil rich granulation tissue enveloped the Ta-silane linkertreated disk, FIG. 9A. With PACPeD treatment, the neutrophils wereabsent with macrophages and matrix making up the bulk of the implantbed, FIG. 9B. After 28 days the control disks had a more pronouncedimplant capsule which was reduced in thickness at PACPeD treated disks,FIG. 10.

Example 22 In Vivo Evaluation of the Inflammatory Response toPolypropylene Admixed with Compound 54

Sample Fiber Preparation

The polypropylene implants for the rat studies were made in a fiberform. After a dry blend was made in the cryo-grinder, the mixture wassubjected to twin screw mixing in a DACA melt mixer. 3 gms of PP and 60mg of Compound 54 (more lipophilic than Compound 38) was used. Theimpact time in the cryogrinder was 5 minutes. The melt mixing chamberwas held at 250° C. The mixing time was 5 minutes with the rpm being 50.No appreciable differences in the torque was seen between the controland the Compound 54 incorporated PP.

A 50 denier fiber with 30% of elongation to break was the target. Theparameters in the DACA melt spin equipment were the following:

Diameter of the spinneret:  0.5 mm Piston speed: 9.82 mm/min Spin speedof the main godet: 1 2.85 RPM Draw ratio: 7 Temperature of the plate:125° C. Temperature of the barrel: 250° C.

The extruded strands from the melt blending were cut into little pieceswhich fed into the barrel more easily. The melt spinning was done at250° C. Because the medical grade polymer degrades after 20 minutes athigh temperature we had to use a flow rate of 0.35 g/min (the amount ofPP in the barrel is 7 g).

Implantation Procedure

Polypropylene fibers, with and without Compound 54 mimic, were implantedsubdermally in 250–300 gram female rats. The polypropylene fiber implantconsisted of a 15 to 20 cm length that was wrapped and tied into afigure eight shape measuring about 2 cm by 0.5. Animals wereanesthetized with a mixture of 50/10 mg/kg Ketamine/Xylazine byintraperitoneal injection. The right flank was shaved and scrubbed withsurgical scrub. A small 1.5 cm long incision was made over the righthaunch. A subcutaneous pocket was made and the appropriate piece ofmaterial was placed in the pocket. The implants were briefly rinsed in70% alcohol and rinsed with 2 dips in sterile saline prior to insertioninto the tissue pocket. The incision was closed with a stainless steelstaple. The rats were returned to their cages for recovery.

The animal were removed from their cages after 21 days post implant andsacrificed by CO₂ inhalation. The implants were removed with overlyingskin attached and fixed in Streck STF fixative overnight at 4–8° C. Theexplants were cut into two or three pieces to expose polymercross-sections and were processed for embedding in paraffin. Routinesections were cut and stained with hematoxylin and eosin or MassonTrichrome and immunostained with an antibody specific of macrophages,EDI (Chemicon Inc.).

In Vivo Response to Implanted Compound 54 Containing Polypropylene

Gross histology examination of control PP fibers attached to the undersurface of the skin flap explants evidenced the fibers to be surroundedby a relatively thick matrix of collagen. The position and overall shapeof the implant were discernible but individual fibers could not be seen.Histological cross-sections confirmed a relatively thick wrap ofconnective tissue. In addition to matrix, higher magnification viewsreveal an intense inflammatory reaction at each fiber. Control fiberscover with one to two layers of cells which appear to be macrophagesbased on positive immunohistochemical staining with the rate macrophagemarker, ED1, FIG. 4A. In addition, foreign body giant cells were alsopresent on all control fibers. These observations are consistent withthe expected chronic inflammatory response.

Compound 54 containing PP fibers exhibited a different response. Grossexamination reveal an implant site in which the individual fibers wereclearly visible. It was obvious that the fibrotic and cellular responsewhich covered the control PP fibers was reduced. Histologically, areduced fibrotic response was apparent, with only a thin wrap of matrixbeing observed in Trichrome stained sections. In addition, theinflammatory response at individual SODm containing fibers was markedlyreduced. Typically, modified fibers were covered by a thin layer ofmatrix and few fibroblasts and only partial coverage by macrophages,FIG. 4B. Foreign body giant cells were seldom observed on modifiedfibers. A count of foreign body giant cells per fiber were performed oncontrol and Compound 54 containing PP fibers. Control fiber FBGC countswere 2.63±1.34 per fiber, n=20 while modified fibers had 1.28±1.04 FBGCper fiber, n=40.

Despite the striking difference in the inflammatory response, the numberor density of fine capillaries appeared to be very similar between thecontrol and modified fibers. This was assessed visually in tissuesspaces between the fibers within the hank and the tissue surrounding thehank implant.

Example 23 Luminol Analysis of Modified Polymers and Metals to DetermineSuperoxide Dismutating Activity

The Michelson assay uses xanthine oxidase and hypoxanthine to producesuperoxide radical anion in situ in a steady-state manner. If noteliminated from the solution with an antioxidant, superoxide then reactswith luminol to produce a measurable amount of light. This reaction isstoichiometric and provides a linear response under pseudo first-orderreaction conditions (i.e. [luminol]>>[O2-]). The light emission ismeasured over several minutes 9as the enzyme-substrate solution producessuperoxide at a specific rate) and the integration of units over thattime is reported. It should then be possible to take samples ofantioxidants and determine the presence of catalyst, the rate ofdismutation, and/or whether the compound is actually catalytic orstoichiometric in its ability to dismute superoxide.

Using this method, we have taken sample films^(1/) p. 74.(lactide/glycolide polymer) doped with Compound 38, a known catalyst forthe dismutation of superoxide and the parent compound in our currentSAR, and analyzed them on a Turner Designs TD-20/20 Luminometer.^(2/)unpublished results. 400 uL of a 0.05 unit/mL xanthine oxidase, 0.1 mMEDTA and 0.1 mM Luminol in 0.1 M glycine buffer at pH 9; 200 uL of a 250uM xanthine solution are added via autoinjector to a one 2 squaremillimeter sample of each film in the sample well. The sample is thenrun on the Luminometer, and the reading translated into an integration.Samples of PEUU surface covalently conjugated with Compound 43 weretested and found to possess superoxide dismutating activity. /Michelson, A. M. In Handbook of Methods of Oxygen Radical Research,Greenwald, R. A., Ed., CRC:Boca Raton, 1989; / Gary W. Franklin;Monsanto Notebook, p. 6136376,

Example 24 Stopped-Flow Kinetic Analysis

Stopped-flow kinetic analysis has been utilized to determine whether acompound can catalyze the dismutation of superoxide (Riley, D. P.,Rivers, W. J. and Weiss, R. H., “Stopped-Flow Kinetic Analysis forMonitoring Superoxide Decay in Aqueous Systems,” Anal. Biochem, 196:344–349 1991). For the attainment of consistent and accuratemeasurements all reagents were biologically clean and metal-free. Toachieve this, all buffers (Calbiochem) were biological grade, metal-freebuffers and were handled with utensils which had been washed first with0.1N HCl, followed by purified water, followed by a rinse in a 10⁻⁴ MEDTA bath at pH 8, followed by a rinse with purified water and dried at65. degree. C. for several hours. Dry DMSO solutions of potassiumsuperoxide (Aldrich) were prepared under a dry, inert atmosphere ofargon in a Vacuum Atmospheres dry glovebox using dried glassware. TheDMSO solutions were prepared immediately before every stopped-flowexperiment. A mortar and pestle were used to grind the yellow solidpotassium superoxide (.about.100 mg). The powder was then ground with afew drops of DMS0 and the slurry transferred to a flask containing anadditional 25 ml of DMSO. The resultant slurry was stirred for ½ h andthen filtered. This procedure gave reproducibly .about.2 mMconcentrations of superoxide in DMSO. These solutions were transferredto a glovebag under nitrogen in sealed vials prior to loading thesyringe under nitrogen. It should be noted that the DMSO/superoxidesolutions are extremely sensitive to water, heat, air, and extraneousmetals. A fresh, pure solution has a very slight yellowish tint.

Water for buffer solutions was delivered from an in-house deionizedwater system to a Barnstead Nanopure Ultrapure Series 550 water systemand then double distilled, first from alkaline potassium permanganateand then from a dilute EDTA solution. For example, a solution containing1.0 g of potassium permanganate, 2 liters of water and additional sodiumhydroxide necessary to bring the pH to 9.0 were added to a 2-liter flaskfitted with a solvent distillation head. This distillation will oxidizeany trace of organic compounds in the water. The final distillation wascarried out under nitrogen in a 2.5-liter flask containing 1500 ml ofwater from the first still and 1.0×10⁻⁶ M EDTA. This step will removeremaining trace metals from the ultrapure water. To prevent EDTA mistfrom volatilizing over the reflux arm to the still head, the 40-cmvertical arm was packed with glass beads and wrapped with insulation.This system produces deoxygenated water that can be measured to have aconductivity of less than 2.0 nanomhos/cm².

The stopped-flow spectrometer system was designed and manufactured byKinetic Instruments Inc. (Ann Arbor, Mich.) and was interfaced to a MACIICX personal computer. The software for the stopped-flow analysis wasprovided by Kinetics Instrument Inc. and was written in QuickBasic withMacAdios drivers. Typical injector volumes (0.10 ml of buffer and 0.006ml of DMSO) were calibrated so that a large excess of water over theDMSO solution were mixed together. The actual ratio was approximately19/1 so that the initial concentration of superoxide in the aqueoussolution was in the range 60–120:M. Since the published extinctioncoefficient of superoxide in H₂O at 245 nm is .about.2250M⁻¹ cm⁻¹ (1),an initial absorbance value of approximately 0.3–0.5 would be expectedfor a 2-cm path length cell, and this was observed experimentally.Aqueous solutions to be mixed with the DMSO solution of superoxide wereprepared using 80 mM concentrations of the Hepes buffer, pH 8.1 (freeacid+Na form). One of the reservoir syringes was filled with 5 ml of theDMSO solution while the other was filled with 5 ml of the aqueous buffersolution. The entire injection block, mixer, and spectrometer cell wereimmersed in a thermostated circulating water bath with a temperature of21EC±0.5EC. Prior to initiating data collection for a superoxide decay,a baseline average was obtained by injecting several shots of the bufferand DMSO solutions into the mixing chamber. These shots were averagedand stored as the baseline. The first shots to be collected during aseries of runs were with aqueous solutions that did not containcatalyst. This assures that each series of trials were free ofcontamination capable of generating first-order superoxide decayprofiles. If the decays observed for several shots of the buffersolution were second-order, solutions of manganese(II) complexes couldbe utilized. In general, the potential SOD catalyst was screened over awide range of concentrations. Since the initial concentration ofsuperoxide upon mixing the DMSO with the aqueous buffer was about 1.2times 10⁻⁴ M, we wanted to use a manganese (II) complex concentrationthat was at least 20 times less than the substrate superoxide.Consequently, we generally screened compounds for superoxide dismutatingactivity using concentrations ranging from 5×10⁻⁷ to 8×10⁻⁶ M. Dataacquired from the experiment was imported into a suitable math program(e.g., Cricket Graph) so that standard kinetic data analyses could beperformed. Catalytic rate constants for dismutation of superoxide bymanganese(II) complexes were determined from linear plots of observedrate constants (k_(obs)) versus the concentration of the manganese(II)complexes. k_(obs) values were obtained from linear plots of lnabsorbance at 245 nm versus time for the dismutation of superoxide bythe manganese(II) complexes.

Example 25 Use of Hyaluronic Acid Esters Surface Covalently Conjugatedwith Compound 43 to Produce a Neural Growth Guide Channel Device

A guide channel with a composite thread/polymeric matrix structurewherein the thread comprises HYAFF 11 (total benzyl ester of HY, 100%esterified) and the matrix is composed of HYAFF 11p75 (benzyl ester ofHY 75% esterified) is obtained by the following procedure.

A. Preparation of Esters

Preparation of the Benzyl Ester of Hyaluronic Acid (HY): 3 g of thepotassium salt of HY with a molecular weight of 162,000 are suspended in200 ml of dimethylsulfoxide; 120 mg of tetrabutylammonium iodide and 2.4g of benzyl bromide are added. The suspension is kept in agitation for48 hours at 30EC. The resulting mixture is slowly poured into 1,000 mlof ethyl acetate under constant agitation. A precipitate is formed whichis filtered and washed four times with 150 ml of ethyl acetate andfinally vacuum dried for twenty four hours at 30EC. 3.1 g of the benzylester product in the title are obtained. Quantitative determination ofthe ester groups is carried out according to the method described onpages 169–172 of Siggia S. and Hanna J. G. “Quantitative organicAnalysis Via Functional Groups,” 4th Edition, John Wiley and Sons.

Preparation of the (Partial) Benzyl Ester of Hyaluronic Acid (HY)—75%Esterified Carboxylic Groups,—25% Salified Carboxylic Groups (Na): 12.4g of HY tetrabutylammonium salt with a molecular weight of 170,000,corresponding to 20 m.Eq. of a monomeric unit, are solubilized in 620 mlof dimethylsulfoxide at 25EC. 120 mg of tetrabutylammonium iodide and15.0 m.Eq. of benzyl bromide are added and the resulting solution iskept at a temperature of 30E for 12 hours. A solution containing 62 mlof water and 9 g of sodium chloride is added and the resulting mixtureis slowly poured into 3,500 ml of acetone under constant agitation. Aprecipitate is formed which is filtered and washed three times with 500ml of acetone/water, 5:1, and three times with acetone, and finallyvacuum dried for eight hours at 30EC.

The product is then dissolved in 550 ml of water containing 1% sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water, 5:1, three times with 500 mlof acetone, and finally vacuum dried for 24 hours at 30EC. 7.9 g of thepartial propyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas Anal. Chem. 33, 1028–1030, (1961).

The HYAFP esters are then surface covalently conjugated with Compound 43as in Example 14.

B. Production of the Device

A thread of total HYAFP 11 esters, 250 denier, with a minimum tensilestrength at break of 1.5 gr/denier and 19% elongation is entwined aroundan electropolished AISI 316 steel bar with an outer diameter of 1.5 mm,which is the desired inner diameter of the composite guide channel. Thewoven product is obtained using a machine with 16 loaders per operativepart.

A typical tube-weaving system system (like the one shown in U.S. Pat.No. 5,879,359) comprising the steel bar with a threaded tube fitted overit is placed in position. The apparatus is rotated at a speed of 115rpm. A quantity of HYAFF 11p75/dimethylsulfoxide solution at aconcentration of 135 mg/ml is spread over the rotating system. Theexcess solution is removed with a spatula, and the system is removedfrom the apparatus and immersed in absolute ethanol. After coagulation,the guide channel is removed from the steel bar and cut to size.

The channel made by the above technique is 20 mm long, 300.mu.m thick,has an internal diameter of 1.5 mm, and has a weight of 40 mg, equal to20 mg/cm.

Example 26 Use of Metals Surface Covalently Conjugated with Compound 43to Produce a Stent

A stent may be formed from surgical stainless steel alloy wire which isbent into a zigzag pattern, and then wound around a central axis in ahelical pattern. Referring now more particularly to FIGS. 11–17, thereis illustrated in FIG. 11 a midpoint in the construction of the stentwhich comprises the preferred embodiment of the present invention. FIG.11 shows a wire bent into an elongated zigzag pattern 5 having aplurality of substantially straight wire sections 9–15 of variouslengths separated by a plurality of bends 8. The wire has first andsecond ends designated as 6 and 7, respectively. Zigzag pattern 5 ispreferably formed from a single strand of stainless steel wire having adiameter in the range of 0.005 to 0.025 inch.

FIG. 13 shows a completed stent 30. The construction of the stent iscompleted by helically winding elongated zigzag pattern 5 about acentral axis 31. Zigzag pattern 5 is wound in such a way that a majorityof the bends 8 are distributed in a helix along the length of the stent30. There are preferably about twelve interconnected bends in eachrevolution of the helix, or six adjacent bends of the zigzag pattern ineach revolution. The construction of stent 30 is completed byinterconnecting adjacent bends of the helix with a filament 32,preferably a nylon monofilament suture. Filament 32 acts as a limitmeans to prevent the stent from further radial expansion beyond thetubular shape shown in FIGS. 13 and 14. The tubular shape has a centralaxis 31, a first end 33 and a second end 35. Each end of stent 30 isdefined by a plurality of end bends 36, which are themselvesinterconnected with a filament 34. Other embodiments of the presentinvention are contemplated in which the end bends 36 are leftunconnected in the finished stent. FIG. 14 shows an end view of stent 30further revealing its tubular shape. FIG. 15 shows stent 30 of FIG. 13when radially compressed about central axis 31 such that the straightwire sections and the bends are tightly packed around central axis 31.

Referring back to FIG. 11, the zigzag pattern is made up of straightwire sections having various lengths which are distributed in a certainpattern to better facilitate the helical structure of the final stentconstruction. For instance, in one embodiment, end wire sections 9 couldbe made to a length of 9 mm followed by two wire sections 11 each being11 mm in length. Wire sections 11 are followed by two 13 mm wiresections 13, which are in turn followed by two wire sections 15 having alength of 15 mm. Sections 15 are followed by a single wire section 17having a length of 17 mm. These gradually increasing wire sections ateither end of the zigzag pattern enable the final stent to have welldefined square ends. In other words, the gradually increasing lengthwire sections on either end of the zigzag pattern enable the final stentto have a tubular shape in which the ends of the tube are substantiallyperpendicular to the central axis of the stent. Following wire section17, there are a plurality of alternating length sections 13 and 15.Short sections 13 being 13 mm in length and long sections 15 being 15 mmin length. This alternating sequence is continued for whatever distanceis desired to correspond to the desired length of the final stent. Thedifference in length between the short sections 13 and long sections 15is primarily dependent upon the desired slope of the helix (see .beta.in FIG. 16) and the desired number of bends in each revolution of thehelix.

FIG. 16 is an enlarged view of a portion of the stent shown in FIG. 13.The body of stent 30 includes a series of alternating short and longsections, 13 and 15 respectively. A bend 8 connects each pair of shortand long sections 13 and 15. Each bend 8 defines an angle 2∀ which canbe bisected by a bisector 40. These short and long sections are arrangedin such a way that bisector 40 is parallel to the central axis 31 of thestent. This allows the stent to be radially compressed withoutunnecessary distortion.

FIG. 12 shows an enlarged view of one end of the zigzag pattern. End 6of the wire is bent to form a closed eye portion 20. Eye 20 ispreferably kept closed by the application of the small amount of solderto the end 6 of the wire after it has been bent into a small loop. Eachof the bends 8 of the zigzag pattern are bent to include a small eyeportion designated as 21 and 23 in FIG. 12, respectively. Eye 21includes a small amount of solder 22 which renders eye 21 closed. Eye 23includes no solder and is left open. The bends 8 which define the helixcan be either in the form of a closed eye, as in eye 21, or open as ineye 23.

After forming the stent, the stent is then modified by surface covalentconjugation with a silyl linker, as in Example 13. By treatment withacid mixtures well known in the art, the stainless steel surface can beoxidized to display a layer of hydroxide. The conjugation then proceedsas in Example 13.

Example 27 Use of Surface Covalently Conjugated Pet Fibers to Produce aWoven Vascular Graft

PET fibers are surface covalently conjugated with Compound 43 accordingto Example 7. The vascular graft fabric is formed from single ply, 50denier, 47 filament (1/50/47) pretexturized, high shrinkage (in excessof approximately 15%), polyethylene terephthalate (PET) yarns woven in aplain weave pattern with 83 ends/inch and 132 picks/inch (prior toprocessing). The vascular graft fabric, prior to processing, has adouble wall size of less than 0.02 inches and preferably has a doublewall thickness of about 0.01 inches. The yarns may be twisted prior toweaving and a graft with 8 twists per inch has provided acceptableproperties. Other weave patterns, yarn sizes (including microdenier) andthread counts also are contemplated so long as the resulting fabric hasthe desired thinness, radial compliance and resistance to long termradial dilation and longitudinal expansion.

The woven fabric is washed at an appropriate temperature, such asbetween 60E–90EC, and then is steam set over a mandrel to provide thedesired tubular configuration. The graft is then dried in an oven or ina conventional dryer at approximately 150EF. Any of the washing,steaming and drying temperatures may be adjusted to affect the amount ofshrinkage of the fabric yarns. In this manner, the prosthetic isradially compliant to the extent necessary for the ends of the graft toconform to the slightly larger anchoring sections of the aorta, butresists radial dilation that otherwise could lead to rupture of theaneurysm and axial extension that could block the entrance to an iliacartery. Radial dilation is considered to occur when a graft expands afurther 5% after radial compliance. The 5% window allows for slightradial expansion due to the inherent stretch in the yarn of the fabric.

The thin walled, woven vascular graft fabric is be formed into a tubularconfiguration and collapsed into a reduced profile for percutaneousdelivery of the prosthetic to the delivery site. The implant issufficiently resilient so that it will revert back to its normal,expanded shape upon deployment either naturally or under the influenceof resilient anchors that secure the implant to the vessel wall, and or,alternatively, struts that prevent compression and twisting of theimplant. The thin wall structure allows small delivery instruments (18Fr or smaller) to be employed when the graft is percutaneously placed.The fine wall thickness also is believed to facilitate the healingprocess. The graft, when used for the repair of an abdominal aorticaneurysm, may be provided in a variety of outer diameters and lengths tomatch the normal range of aortic dimensions.

The biologically compatible prosthetic fabric encourages tissue ingrowthand the formation of a neointima lining along the interior surface ofthe graft, preventing clotting of blood within the lumen of theprosthetic which could occlude the graft. The graft has sufficientstrength to maintain the patency of the vessel lumen and sufficientburst resistance to conduct blood flow at the pressures encountered inthe aorta without rupturing. The graft is usually preclotted with eitherthe patient's own blood or by coating the fabric with an imperviousmaterial such as albumin, collagen or gelatin to prevent hemorrhaging asblood initially flows through the graft. Although a constant diametergraft is preferred, a varying dimensioned prosthetic also iscontemplated. The graft is also usually provided with one or moreradiopaque stripes to facilitate fluoroscopic or X-ray observation ofthe graft.

Example 28 Use of Copolymerized Polyurethane to to Insulate CardiacSimulator Lead Wire

A die-clad composite conductor is made with a highly conducting core anda cladding layer. Copper and copper alloys are particularly suitable forthe core material of the composite conductor. Pure copper is preferable,but alloys such as Cu0.15Zr, Cu4Ti, Cu2Be, Cu1.7Be, Cu0.7Be, Cu28Zn,Cu37Zn, Cu6Sn, Cu8Sn and Cu2Fe may be used. A metal selected from thegroup consisting of tantalum, titanium, zirconium, niobium,titanium-base alloys, platinum, platinum-iridium alloys,platinum-palladium alloys and platinum-rhodium alloys is applied as acladding layer to the conducting core by drawing through a die. Thecladding layer thickness is between 0.0025 and 0.035 mm, while the corediameter is between 0.04 and 0.03 mm. Although a single strand conductorcould be used, the risks of breakage are reduced and the conductivity isincreased without going beyond the above described preferred ranges forcore diameter and cladding if a stranded conductor is used. Furthermorea stranded conductor provides increased flexibility. Thus, it ispreferred that the conductor in the cable be composed of two or morethinner strands twisted together.

The clad wire conductor is enclosed in an elastic covering tube, whichconsists of a synthetic elastomer such as flexible polyurethane. Apolyurethane which has been copolymerized with a PACPeD catalyst, suchas the polyurethane of Example 12, should be used. It is sufficientlyelastic and flexible to make possible its introduction into the heartchamber simply by being carried along through the blood stream. Thebiocompatability of the clad wire conductor of the cable is improved byoxidizing the surface of the clad wire and covalently conjugating aPACPeD catalyst to the wire, as in Example 13.

1. A modified biopolymer comprising a biopolymer chosen from the groupconsisting of chitin, chitosan, cellulose, methyl cellulose, hyaluronicacid, keratin, fibroin, collagen, elastin, and saccharide polymersattached to at least one non-proteinaceous catalyst capable ofdismutating superoxide in the biological system or precursor ligandthereof, wherein the non-proteinaceous catalyst capable of dismutatingsuperoxide is chosen from the group consisting of:


2. Modified hyaluronic acid prepared by reaction of hyaluronic acid witha non-proteinaceous catalyst capable of dismutating superoxide havingthe structure:

or a precursor ligand thereof under conditions appropriate to covalentlyattach the non-proteinaceous catalyst capable of dismutating superoxideto the hyaluronic acid.
 3. The modified biopolymer of claim 1, whereinthe biopolymer is hyaluronic acid.
 4. A modified biopolymer comprisinghyaluronic acid attached to at least one catalyst having the structure:


5. A modified biopolymer comprising hyaluronic acid attached to at leastone catalyst having the structure:


6. A modified biopolymer comprising hyaluronic acid attached to at leastone catalyst having the structure: